SECTION I: BIOCHEMICAL AND MOLECULAR CONCEPTS

INTRODUCTION

Chemistry is the science of matter; it encompasses the structure, physical properties, and reactivities of atoms and their compounds. In many respects, toxicology is the science of the interactions of matter with living entities; chemistry and toxicology are therefore intimately linked. The study of the principles of inorganic, organic, and biologic chemistry offers important insight into the mechanisms and clinical manifestations of xenobiotics and poisoning.⁵ This chapter reviews many of these tenets and provides relevance to the current practice of medical toxicology.

THE STRUCTURE OF MATTER

Basic Structure

Matter includes the substances of which everything is made. Elements are the foundation of matter, and all matter is made from one or more of the known elements. An atom is the smallest quantity of a given element that retains the properties of that element. Atoms consist of a nucleus, incorporating protons and neutrons, coupled with its orbiting electrons. The atomic number is the number of protons in the nucleus of an atom, and it is a whole number that is unique for each element. Thus, elements with 6 protons are always carbon, and all forms of carbon have exactly 6 protons. However, although the vast majority of carbon nuclei have 6 neutrons in addition to the protons, accounting for an atomic mass (protons plus neutrons) of 12 (¹²C), a small proportion of naturally occurring carbon nuclei, called isotopes, have 8 neutrons and a mass number of 14 (¹⁴C). This is the reason that the atomic weight of carbon displayed on the periodic table is 12.011, and not 12, as it actually represents the average atomic masses of all isotopes found in nature weighted by their frequency of occurrence. ¹⁴C is also a radioisotope, which is an isotope with an unstable nucleus that emits radiation until it achieves a stable state (Chap. 128). The atomic weight, measured in grams per mole (g/mol), also indicates the molar mass of the element. That is, in one atomic weight of matter (12.011 g for carbon) there is one mole (6.022140857 × 10²³) of atoms as defined by Avogadro’s constant.

Elements combine chemically to form compounds, which generally have physical and chemical properties that differ from those of the constituent elements. The elements in a compound can be separated only by chemical means that destroy the original compound, as occurs during the burning (ie, oxidation) of a hydrocarbon, a process that releases the carbon principally as carbon dioxide and the hydrogen principally as water (H₂O). This important property differentiates compounds from mixtures. Mixtures are combinations of elements or compounds that can be separated by physical means, such as the distillation of petroleum into its hydrocarbon components or the evaporation of seawater to separate water from sodium chloride and other substances. With notable exceptions, such as the elemental forms of many metals or halogens (eg, Cl₂), most xenobiotics are compounds or mixtures.

Dmitri Mendeleev, a Russian chemist in the mid-19th century, recognized that when all of the known elements were arranged in order of atomic weight, certain patterns of reactivity became apparent. The result of his work was the Periodic Table of the Elements (Fig. 10–1), which, with some minor alterations, is still an essential tool today. All of the currently recognized elements are represented; those heavier than uranium do not occur in nature. Many of the symbols used to identify the elements refer to the Latin name of the element. For example, silver is Ag, for argentum, and mercury is Hg, for hydrargyrum, literally “silver water.”

The periodicity of the table relates to the electrons that circle the nucleus in discrete orbitals. Although the details of quantum mechanics and electronic configuration are complex, it is important to review some aspects in order to predict chemical reactivity. Orbitals, or quantum shells, represent the energy levels in which electrons exist around the nucleus. The orbitals are identified by various schemes, but the maximum number of electrons each orbital can contain is calculated as 2x², where x represents the numerical rank order of the orbital. Thus, the first orbital may contain 2 electrons, the second orbital may contain 8, the third may contain 18, and so on. However, the outermost shell (designated by s, p, d, f nomenclature) of each orbital may only contain up to 8 electrons. This is irrelevant through element 20, calcium, because there is no need to fill the third-level or d shells. Even though the third orbital may contain 18 electrons, once 8 are present the 4s electrons dip below the 3d electrons in energy and this shell begins to fill. This occurs at element 21, scandium, and accounts for its chemical properties and those of the other transition elements. Transition elements are chemically defined as elements that form at least one ion with a partially filled subshell of d electrons. The inert gas elements, which are also known as noble gases, have complete outermost orbitals, accounting for their lack of reactivity under standard conditions.

In general, only electrons in unfilled shells, or valence shells, are involved in chemical reactions. This property relates to the fact that the most stable form of an element occurs when the configuration of its valence shell resembles that of the nearest noble gas, found in group 18 on the periodic table. This state can be obtained through the gaining, losing, or sharing of electrons with other elements and is the basis for virtually all chemical reactions.

INORGANIC CHEMISTRY

The Periodic Table

Chemical Reactivity

Broadly, the periodic table is divided into metals and nonmetals. Metals, in their elemental form, are typically malleable solids that conduct electricity, whereas nonmetals are usually dull, fragile, nonconductive compounds (C, N, P, O, S, Se, and the halogens). Metals are found on the left side of the periodic table, and account for the majority of the elements, whereas nonmetals are on the right side. Separating the 2 groups are the metalloids, which fall on a jagged line starting with boron (B, Si, Ge, As, Sb, Te, At). Metalloids have chemical properties that are intermediate between the metals and the nonmetals. Each column of elements is termed a family or group, and each row is a period. Although the table is conceived and organized in periods, trends in the chemical reactivity, and therefore toxicity, typically exist within the groups.

In 1990, the International Union of Pure and Applied Chemistry (IUPAC) replaced the existing numbering schemes for the periodic table columns with a unified and simplified nomenclature. Using this format, which is included in this chapter, the groups are simply incrementally numbered from 1 to 18 from left to right.²

The ability of any particular element to produce toxicologic effects relates directly to one or more of its many physicochemical properties. These properties will, to some extent, be predicted by their location on the periodic table. For example, arsenate will substitute for phosphate in the mitochondrial production of adenosine triphosphate (ATP), creating adenosine diphosphate monoarsenate (Chap. 11). Because this compound is unstable and not useful as an energy source, energy production by the cell fails; in this manner, arsenic interferes with oxidative phosphorylation. The existence of an interrelationship between Ca²⁺ and either Mg²⁺ or Ba²⁺ is predictable, although the actual effects are not. Under most circumstances, Mg²⁺ is a Ca²⁺ antagonist, and patients with hypermagnesemia present with neuromuscular weakness caused by the blockade of myocyte calcium channels. Alternatively, Ba²⁺ mimics Ca²⁺ and closes Ca²⁺-dependent K⁺ channels in myocytes, producing life-threatening hypokalemia. The physiologic relationships among the ions of lithium (Li⁺), potassium (K⁺), and sodium (Na⁺) are also consistent with their chemical similarities (all alkali metals in Group IA). However, the clinical similarity between thallium (thallous) ion (Tl⁺) and K⁺ is not immediately predictable. Other than their monovalent nature (ie, 1+ charge), it is difficult to predict the substitution of Tl⁺ (Group 13, Period 6) for K⁺ (Group 1, Period 4) in membrane ion channel functions, until the similarity of their ionic radii is known (Tl⁺, 1.47 Å; K⁺, 1.33 Å).

An emerging physicochemical property of toxicological interest is particle size. The chemical and toxicologic properties of a given element or molecule change as the particle size is reduced. An entire field of nanotoxicology has emerged to study the adverse health effects of nanoparticles, which are ultrafinely divided particles, ranging from approximately 1 to 100 nm in diameter. Differences in absorption, biodistribution, and structure-activity effects, for example, enhance the toxicologic potential of otherwise nontoxic xenobiotics.⁷

Alkali and Alkaline Earth Metals

Alkali metals (Group 1, formerly Group IA: Li, Na, K, Rb, Cs, Fr) and hydrogen (not an alkali metal on Earth) have a single outer valence electron and lose this electron easily to form compounds with a valence of 1+. The alkaline earth metals (Group 2, formerly Group IIA: Be, Mg, Ca, Sr, Ba, Ra) are located between the alkali and rare earth (Group 3, formerly Group IIIB), readily lose 2 electrons, and form cations with a 2+ charge. In their metallic form, members of both of these groups react violently with water to liberate strongly basic solutions, accounting for the word alkali in their group names (2Na⁰ + 2H₂O → 2NaOH + H₂).

The soluble ionic forms of sodium, potassium, or calcium, which are critical to survival, also produce life-threatening symptoms following excessive intake (Chap. 12). Xenobiotics interfere with the physiologic role of these key electrolytes. Li⁺ mimics K⁺ after entering neurons through K⁺ channels, and serves as an inadequate substrate for the repolarizing of Na⁺,K⁺-ATPase. Li⁺ thus interferes with cellular K⁺ homeostasis and alters neuronal repolarization, accounting for the neuroexcitability manifesting as tremor. Similarly, as noted previously, the molecular effects of Mg²⁺ and Ba²⁺ supplant those of Ca²⁺.

More commonly, the consequential toxicities ascribed to alkali or alkaline earth salts relate to the anionic component. In the case of NaOH or Ca(OH)₂, it is the hydroxide anion, whereas with potassium cyanide (KCN) it is the cyanide (CN^–) anion. When the chemical reactivity of an ionic compound (including its cellular toxicity) can be ascribed solely to one of the ions, any other ion in the compound is referred to as a spectator ion.

Transition Metals

Unlike the alkali and alkaline earth metals, most other metallic elements are neither soluble nor reactive. This includes the transition metals (Groups 4-12; formerly Groups IB to VIIB, VIII, IB and IIB), a large group that contains several ubiquitous metals such as iron (Fe) and copper (Cu). These elements, in their metallic form, are widely used in both industrial and household applications because of their high tensile strength, density, and melting point, which is partly a result of their ability to delocalize the electrons in the d orbital throughout the metallic lattice. Transition metals also form brightly colored salts that find widespread applications, including as pigments for paints or fireworks. The ionic forms of these elements are of greater toxicologic importance than their metallic/elemental forms. Chemically, transition elements form at least one ion with a partially filled subshell of d electrons. Because the transition metals have partially filled valence shells, they are capable of obtaining several, usually positive, oxidation states. This important mechanism explains the role of transition metals in oxidation/reduction (redox) reactions, generally as electron acceptors (see Reduction-Oxidation below). This reactivity is used by organisms in various physiologic, catalytic, and coordination roles, such as at the active sites of enzymes and in hemoglobin. As expected, the substantial reactivity of these transition metal elements is associated with cellular injury caused by several mechanisms, including the generation of reactive oxygen species (Fig. 10–2). Manganese ion, as an example, is implicated in free radical damage of the basal ganglia, causing parkinsonism.

Heavy Metals

Heavy metal is often loosely used to describe all metals of toxicologic significance, but in reality the term should be reserved to describe only those metals in the lower period of the periodic table, particularly those with atomic masses greater than 200 Da. The chemical properties and toxicologic predilection of these elements vary, but a unifying toxicologic mechanism is electrophilic interference with nucleophilic sulfhydryl-containing enzymes. Some of the heavy metals also participate in the generation of free radicals through Fenton chemistry (Fig. 10–2). The likely determinant of the specific toxicologic effects produced by each metal is the tropism for various physiologic systems, enzymes, or microenvironments; thus, lipophilicity, water solubility, ionic size, and other physicochemical parameters are undoubtedly critical. Also, because the chemistry of metals varies dramatically based on the chemical form (ie, organic, inorganic, or elemental), as well as the charge on the metal ion, prediction of the clinical effects of a particular metal is often difficult.

Mercury

Elemental mercury (Hg⁰) is unique in that it is the only metal that exists in liquid form at room temperatures. As such, it is capable of creating semisolid solutions, or amalgams, with other metals. Although relatively innocuous if ingested as a liquid, elemental mercury is readily volatilized (as a result of its high vapor pressure), transforming it into a significant pulmonary mucosal irritant upon inhalation. Pulmonary exposure increases its systemic bioavailability. Elemental mercury undergoes biotransformation in the erythrocyte and brain to the mercuric (Hg²⁺) form, which has a high affinity for sulfhydryl-containing molecules, such as proteins. This causes a depletion of glutathione in organs such as the kidney, and also initiates lipid peroxidation. The mercurous form (Hg⁺) is considerably less toxic than the mercuric (Hg²⁺) form, perhaps because of its reduced water solubility, which limits absorption. Organic mercurial compounds, such as methylmercury and dimethylmercury, are formed environmentally by anaerobic bacteria containing the methylating compound methylcobalamin, a vitamin B₁₂ analog (Chap. 95).

Thallium

Metallic thallium (Tl⁰) is used in the production of electronic equipment, and is itself minimally toxic. Thallium ions, however, have physicochemical properties that closely mimic potassium ions, allowing them to participate in and often alter the myriad physiologic activities related to potassium. This property is exploited during a thallium-stress test to assess for myocardial ischemia or infarction; because ischemic myocardial cells lack adequate energy for normal Na⁺,K⁺-ATPase function, they cannot exchange sodium for potassium (or radioactive thallium administered during a stress test). This produces a “cold spot” in the ischemic areas on cardiac scintigraphy (Chap. 99).

Lead

Lead is not abundant in the Earth’s crust (0.002%), but mining and industrial use make it a significant toxicologic concern. Lead exposure occurs during the smelting process or from one of its diverse commercial applications. Most of the useful lead compounds are inorganic plumbous (Pb²⁺) salts, but plumbic (Pb⁴⁺) compounds are also used. The Pb²⁺ compounds are typically ionizable, releasing Pb²⁺ when dissolved in a solvent, such as water. Lead ions are absorbed in place of Ca²⁺ ions by the gastrointestinal tract and replace Ca²⁺ in certain physiologic processes. This mechanism is implicated in the neurotoxic effect of lead ions. Lead compounds tend to be covalent compounds that do not ionize in water. Some Pb⁴⁺ compounds are oxidants. Although elemental lead is not itself toxic, it rapidly develops a coating of toxic lead oxide or lead carbonate on exposure to air or water (Chap. 93).

Metalloids

Although the metalloids (B, Si, Ge, As, Sb, Te, At) share many physical properties with metals, they differ in their propensity to form compounds with both metals and the nonmetals carbon, nitrogen, or oxygen. Metalloids are either oxidized or reduced in chemical reactions.

Arsenic

Toxicologically important inorganic arsenic compounds exist in either the pentavalent arsenite (As⁵⁺) form or the trivalent arsenate (As³⁺) form. The reduced water solubility of arsenate compounds (such as arsenic pentoxide) accounts for its limited clinical toxicity when compared to trivalent arsenic trioxide. The trivalent form of arsenic is primarily a nucleophilic toxin, binding sulfhydryl groups and interfering with enzymatic function (Chaps. 11 and 86).

Nonmetals

The nonmetals (C, N, P, O, S, Se, halogens) are highly electronegative and are toxic in either their compounded or their elemental form. The nonmetals with large electronegativity, such as O₂ or Cl₂, generally oxidize other elements in chemical reactions. Those with lesser electronegativity, such as C, behave as reducing agents.

Halogens

In their highly reactive elemental form, which is a covalent dimer of halogen atoms, the halogens (F, Cl, Br, I, At) carry the suffix ine (eg, Cl₂, chlorine). Halogens require the addition of one electron to complete their valence shell, and are strong oxidizing agents. Because they are highly electronegative, they readily form halides (eg, Cl^–, chloride) by abstracting electrons from less electronegative elements. Thus, the halogen ions, in their stable ionic form, generally carry a charge of –1. The halides, although much less reactive than their respective elemental forms, are reducing agents. The hydrogen halides (eg, HCl, hydrogen chloride) are gases under standard conditions, but they ionize when dissolved in aqueous solution to form hydrohalidic acids (eg, HCl, hydrochloric acid). All hydrogen halides except HF (hydrogen fluoride) ionize nearly completely in water to release H⁺ and are considered strong acids. Because of its small ionic radius, lack of charge dispersion, and the intense electronegativity of the fluorine atom, HF ionizes poorly and is a weak acid. This specific property of HF has important toxicologic implications (Chap. 104).

Group 18 (formerly Group VIIIA): Inert Gases. Inert gases (He, Ne, Ar, Kr, Xe, Rn), also known as noble gases, maintain completed valence shells and are thus unreactive except under extreme experimental conditions. Despite their lack of chemical reactivity, the inert gases are toxicologically important as simple asphyxiants, causing hypoxia if they displace ambient oxygen from a confined space (Chap. 121). During high-concentration exposure, inert gases produce anesthesia, and xenon is used as an anesthetic agent. Radon, although chemically unreactive, is radioactive, and prolonged exposure is associated with the development of lung cancer.

Bonds

Electrons are not generally shared evenly between atoms when they form a compound unless the bond is between the same elements, as in Cl₂. When different elements are involved, one will exert a larger attraction for the shared electrons. The degree to which an element draws the shared electron is determined by the electronegativity of the element (Fig. 10–3). The electronegativity of each element was catalogued by Linus Pauling and relates to the ionic radius, or the distance between the orbiting electron and the nucleus, and the shielding effects of the inner electrons. Electronegativity rises toward the right of the periodic table, corresponding with the expected charge obtained on an element when it forms a bond. Fluorine has the highest electronegativity of all elements, which explains many of the toxicologic properties of fluorine.

Several types of bonds exist between elements when they form compounds. When one element gains valence electrons and another loses them, the resulting elements are charged and attract one another in an ionic, or electrovalent, bond. An example is sodium chloride (NaCl), or table salt, in which the electronegativity difference between the elements is 1.9, or greater than the electronegativity of the sodium (Fig. 10–3). Thus, the chloride wrests control of the electrons in this bond. In solid form, ionic compounds exist in a crystalline lattice, but when put into solution, as in water or in blood (serum), the elements separate and form charged particles, or ions (Na⁺ and Cl^–). The ions are stable in solution because their valence shells contain 8 electrons and are complete. The properties of ions differ from both the original atom from which the ion is derived and the noble gas with which it shares electronic structure.

It is important to recognize that when a mole of a salt, such as NaCl (molecular weight 58.45 g/mol), is put in aqueous solution, 2 moles of particles result. This is because NaCl is essentially fully ionized in water; that is, it produces one mole of Na⁺ (23 g/mol) and one mole of Cl^– (35.5 g/mol). For salts that do not ionize completely, less than the intrinsic number of moles are released and the actual quantity liberated can be predicted based on the defined solubility of the compound, or the solubility product constant (K_sp). For ions that carry more than a single charge, the term equivalent is often used to denote the number of moles of other particles to which one mole of the substance will bind. Thus, one equivalent of calcium ions will typically bind 2 moles (or equivalents) of chloride ions (which are monovalent) because calcium ions are divalent. Alternatively stated, a 10% calcium chloride (CaCl₂) aqueous solution contains approximately 1.4 mEq/mL or 0.7 mmol/mL of Ca²⁺.

Compounds formed by 2 elements of similar electronegativity have little ionic character because there is little impetus for separation of charge. Instead, these elements share pairs of valence electrons, a process known as covalence. The resultant molecule contains a covalent bond, which is typically very strong and generally requires a high-energy chemical reaction to disrupt it. There is wide variation in the extent to which the electrons are shared between the participants of a covalent bond, and the physicochemical and toxicologic properties of any particular molecule are in part determined by its nature. Sharing is rarely symmetric (as in oxygen {O₂} or chlorine {Cl₂}). When electrons are shared asymmetrically (localized to a greater degree around one of the component atoms), the bond is called polar. Importantly, however, the presence of polar bonds does not mean that the compound itself is polar. For example, methane contains a carbon atom that shares its valence electrons with 4 hydrogen atoms, in which there is a small charge separation between the elements (electronegativity {EN} difference = 0.40). Although each of the carbon hydrogen bonds is polar, however, the tetrahedral configuration of the molecule means that the asymmetries cancel each other out, resulting in no net polarity, rendering the compound nonpolar. The lack of polarity demonstrates that methane molecules have little affinity for other methane molecules and they are held together only by weak intermolecular bonds. This explains why methane is highly volatile and therefore exists as a gas under standard temperature and pressure.

Because the EN differences between hydrogen (EN = 2.20) and oxygen (EN = 3.44) are significant (EN difference = 1.24), the electrons in the hydrogen-oxygen bonds in water are drawn toward the oxygen atom, giving it a partial negative charge and the hydrogen a partial positive charge. Because H₂O is angular, not linear or symmetric, water is a polar molecule. Water molecules are held together by hydrogen bonds, which are bonds between electropositive hydrogen atoms and the strongly electronegative oxygen atoms. Hydrogen bonds are stronger than other intermolecular bonds (eg, van der Waals forces; see later). Hydrogen bonds have sufficient energy to open many ionic bonds and solvate ions (ie, cause ionic compounds to go into solution). In this process, the polar ends of the water molecule surround the charged particles of the dissolved salt. As one corollary, nonpolar molecules are relatively insoluble in polar compounds such as water, as there is little similarity between the nonpolar methane and the polar water molecules. As a second corollary, salts cannot be solvated by nonpolar compounds, and thus a salt, such as sodium chloride, has almost no solubility in a nonpolar solvent such as carbon tetrachloride.

Alternatively, the stability and irreversibility of the bond between an organic phosphorus compound and the cholinesterase enzyme are a result of covalent phosphorylation of an amino acid at the active site of the enzyme. The resulting bond is essentially irreversible in the absence of another chemical reaction (Fig. 110–3).

Compounds often share multiple pairs of electrons. For example, the 2 carbon atoms in acetylene (HC≡CH) share 3 pairs of electrons between them, and each shares one pair with a hydrogen. Carbon and nitrogen share 3 pairs of electrons in forming cyanide (C≡N^–), making this bond very stable and accounting for the large number of xenobiotics capable of liberating cyanide. Complex ions are covalently bonded groups of elements that behave as a single element. For example, hydroxide (OH^–) and sulfate (SO₄^2–) form sodium salts as if they were simply the ion of a single element (such as chloride).

Noncovalent bonds, such as hydrogen or ionic bonds, are important in the interaction between ligands and receptors as well as between ion channels and enzymes. These are low-energy bonds and easily broken. Van der Waals forces, also known as London dispersion forces, are intermolecular forces that arise from induced dipoles as a consequence of nonuniform distribution of the molecular electron cloud. These forces become stronger as the atom (or molecule) becomes larger because of the increased polarizability of the larger, more dispersed electron clouds. This accounts for the fact that under standard temperature and pressure, fluorine and chlorine are gases, whereas bromine is a liquid, and iodine is a solid.

Reduction-Oxidation

Reduction-oxidation (redox) reactions involve the movement of electrons from one atom or molecule to another, and comprise 2 interdependent reactions (reduction and oxidation). Reduction is the gain of electrons by an atom that is thereby reduced. The electrons derive from a reducing agent, which in the process becomes oxidized. Oxidation is the loss of electrons from an atom, which is, accordingly, oxidized. An oxidizing agent accepts electrons and, in the process, is reduced. By definition, these chemical reactions involve a change in the valence of an atom. It is also important to note that acid-base and electrolyte chemical reactions involve electrical charge interactions but no change in valence of any of the involved components. The implications of redox chemistry for toxicology are significant. For example, the oxidation of ferrous (Fe²⁺) to ferric (Fe³⁺) iron within the hemoglobin molecule creates the dysfunctional methemoglobin molecule (Chap. 124).

The oxidation state of an atom or molecule plays an important role in its toxicity. Elemental lead and mercury are both intrinsically harmless metals, but when oxidized to their cationic forms produce devastating clinical effects. The metabolism of ethanol to acetaldehyde involves a change in the oxidation state of the molecule. In this case, an enzyme, alcohol dehydrogenase, oxidizes (ie, removes electrons from) the C-O bond and delivers the electrons to oxidized nicotinamide adenine dinucleotide (NAD⁺), reducing it to NADH. As in this last example, oxidation is occasionally used to signify the gain of oxygen by a substance. That is, when elemental iron (Fe⁰) undergoes rusting to iron oxide (Fe₂O₃), it is said to oxidize. The use of this term is consistent because in the process of oxidation, oxygen derives electrons from the atom with which it is reacting and combining.

Reactive Oxygen Species

Free radicals are reactive molecules that contain one or more unpaired electrons. They are typically neutral but also can be anionic or cationic. However, because certain toxicologically important reactive molecules, such as hydrogen peroxide (H₂O₂) and ozone (O₃), do not contain unpaired electrons, the term reactive species is preferred. The reactivity of these molecules directly relates to their attempts to fill their outermost orbitals by receiving an electron; the result is oxidative stress on the biologic system. Molecular oxygen is actually a diradical with 2 unpaired electrons in the outer orbitals. However, its reactivity is less than that of the other radicals because the unpaired electrons have parallel spins, so catalysts (ie, enzymes or metals) are typically involved in the use of oxygen in biologic processes.

Reactive species are continuously generated as a consequence of endogenous metabolism, and there is an efficient detoxification system for their control. However, under conditions of either excessive endogenous generation or exposure to exogenous reactive species, the physiologic defenses against these toxic products are overwhelmed. When this occurs, reactive species induce direct cellular damage and initiate a cascade of additional harmful oxidative reactions.⁴

Intracellular organelles, particularly the mitochondria, are damaged by various reactive species. This causes further injury to the cell as energy failure occurs. This initial damage is exacerbated by the activation of the host inflammatory response by chemokines that are released from cells in response to reactive species–induced damage. This inflammatory response aggravates cellular damage. The resultant membrane dysfunction or damage causes cellular apoptosis or necrosis.

The most important reactive oxygen species in medical toxicology are derived from oxygen, although those derived from nitrogen are also important. Table 10–1 lists some of the important reactive oxygen and nitrogen species.

The biradical nature of oxygen explains both the physiologic and toxicologic importance of oxygen in biologic systems. Physiologically, the majority of oxygen used by the body serves as the ultimate electron acceptor in the mitochondrial electron transport chain. In this situation, 4 electrons are added to each molecule of oxygen to form 2 water molecules (O₂ + 4H⁺ + 4e^– → 2H₂O).

Superoxide (O₂^–) is “mutated” from oxygen within neutrophil and macrophage lysosomes as part of the oxidative burst, which serves to help eliminate infectious agents and damaged cells. Superoxide is subsequently enzymatically converted, or “dismutated,” into hydrogen peroxide by superoxide dismutase (SOD). Hydrogen peroxide also can subsequently converted into hypochlorous acid by the enzymatic addition of chloride by myeloperoxidase. Both hydrogen peroxide and hypochlorite ion are more potent reactive oxygen species than superoxide. However, this lysosomal protective system is also responsible for tissue damage following poisoning as the innate inflammatory response attacks xenobiotic-damaged cells. Examples include acetaminophen (APAP)-induced hepatotoxicity (Chap. 33), carbon monoxide neurotoxicity (Chap. 122), and chlorine-induced pulmonary toxicity (Chap. 121), each of which is altered, at least in experimental systems, by the addition of scavengers of reactive species.

Although superoxide and hydrogen peroxide are reactive species, it is their conversion into the hydroxyl radical (OH·) that accounts for their most consequential effects. The hydroxyl radical is generated by the Fenton reaction (Fig. 10–2), in which hydrogen peroxide is decomposed in the presence of a transition metal.³ This catalysis typically involves Fe²⁺, Cu⁺, Cd²⁺, Cr⁵⁺, Ni²⁺, or Mn²⁺. The Haber-Weiss reaction (Fig. 10–2), in which a transition metal catalyzes the combination of superoxide and hydrogen peroxide, is another important means of generating the hydroxyl radical. Alternatively, superoxide dismutase, within the erythrocyte, contains an ion of copper (Cu²⁺) that participates in the catalytic reduction of superoxide to hydrogen peroxide and the subsequent detoxification of hydrogen peroxide by glutathione peroxidase or catalase.

Transition metal cations frequently bind to the cellular nucleus and locally generate reactive oxygen species, most importantly hydroxyl radicals. This results in DNA strand breaks and modification, accounting for the mutagenic effects of many transition metals. In addition to the important role that transition metal chemistry plays following iron or copper salt poisoning, the long-term consequences of chronic transition metal poisoning are exemplified by asbestos. The iron contained in asbestos serves as the source of the Fenton-generated hydroxyl radicals that are responsible for the pulmonary fibrosis and cancers associated with long-term exposure.

The most important toxicologic effects of reactive oxygen species occur on the cell membrane, and are caused by the initiation by hydroxyl radicals of the lipid peroxidative cascade. The alteration of these lipid membranes ultimately causes membrane destruction. Identification of released oxidative products such as malondialdehyde is a common method of assessing lipid peroxidation.

Under normal conditions, there is a delicate balance between the formation and immediate detoxification of reactive oxygen species. For example, the conversion of the superoxide radical to hydrogen peroxide via SOD is rapidly followed by the transformation of hydrogen peroxide to water by glutathione peroxidase or catalase. The fact that transition metals exist in “free” form in only minute quantities in biologic systems minimizes the formation of hydroxyl radicals; that is, cells have developed extensive systems by which transition metal ions are sequestered and rendered harmless. Ferritin (binds iron), ceruloplasmin (binds copper), and metallothionein (binds cadmium) are all specialized proteins that safely sequester transition metal ions. Certain proteins and enzymes such as hemoglobin or SOD have critical biological functions associated with the transition metals at their active sites.

Detoxification of certain reactive species is difficult because of their extreme reactivity. Widespread antioxidant systems typically scavenge reactive species before tissue damage occurs. An example is glutathione, a reducing agent and nucleophile, which prevents both exogenous oxidants from producing hemolysis and the APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI) from damaging the hepatocyte (Chap. 33).

The key reactive nitrogen species is nitric oxide. At typical physiologic concentrations, this radical is responsible for vascular endothelial relaxation through stimulation of guanylate cyclase. However, during oxidative burst, high concentrations of nitric oxide are formed from L-arginine. At these concentrations, nitric oxide is directly damaging to tissue and also reacts with the superoxide radical to generate the peroxynitrite anion. This is particularly important because peroxynitrite spontaneously degrades to form the hydroxyl radical. Peroxynitrite ion is implicated in both the delayed neurologic effects of carbon monoxide poisoning and the hepatic injury from APAP.

Redox Cycling

Although transition metals are an important source of reactive species, certain xenobiotics are also capable of independently generating reactive species. Most do so through a process called redox cycling, in which a molecule accepts an electron from a reducing agent and subsequently transfers that electron to oxygen, generating the superoxide radical. At the same time, this second reaction regenerates the parent molecule, which itself can gain another electron and restart the process. The toxicity of paraquat (Chap. 109) is selectively localized to pulmonary endothelial cells. Its pulmonary toxicity results from redox cycling generation of reactive oxygen species (Fig. 109–3). A similar process, localized to the heart, occurs with anthracycline antineoplastics such as doxorubicin.

Acid-Base Chemistry

Water is amphoteric, which means that it can function as either an acid or a base, much the same way as the bicarbonate ion (HCO₃^–). Because of the amphoteric nature of water, H⁺, despite the nomenclature, does not ever actually exist in aqueous solution; rather, it is covalently bound to a molecule of water to form the hydronium ion (H₃O⁺). However, the term H⁺, or proton, is used for convenience.

Even in a neutral solution, a tiny proportion of water is always undergoing ionization to form both H⁺ and OH^– in exactly equal amounts. By convention, however, it is the quantity of H⁺ that is measured to determine the acidity or alkalinity of the solution. The units for this measurement are pH, which is –log[H⁺]. In a perfect system at equilibrium, the concentration of H⁺ ions in water is precisely 0.0000001, or 10^–7, mol/L and that of OH^– is the same. The number of H⁺ ions increases when an acid is added to the solution and falls when an alkali is added. The negative log of 10^–7 is 7, and the pH of a neutral aqueous solution is thus 7. In actuality, the pH of water is approximately 6 because of dissolution of ambient carbon dioxide to form carbonic acid (H₂O + CO₂ → H₂CO₃), which ionizes to form H⁺ and bicarbonate (HCO₃^–).

There are many definitions of acid and base. The 3 commonly used definitions are those advanced by (1) Svante Arrhenius, (2) Brønsted and Lowry, and (3) Lewis. Because our focus is on physiologic systems, which are most commonly aqueous solutions, the original definition by the Swedish chemist Arrhenius is the most practical. In this view, an acid releases hydrogen ions, or protons (H⁺), in water. Conversely, a base produces hydroxyl ions (OH^–) in water. Thus, hydrogen chloride (HCl), a neutral gas under standard conditions, dissolves in water to liberate H⁺ and is therefore an acid.

For nonaqueous solutions, the Brønsted-Lowry definition is preferable. An acid, in this schema, is a substance that donates a proton and a base is one that accepts a proton. Thus, any molecule that has a hydrogen atom in the 1+ oxidation state is technically an acid, and any molecule with an unbound pair of valence electrons is a base. Because most of the acids or bases of toxicologic interest have ionizable protons or available electrons, respectively, the Brønsted-Lowry definition is most often considered when discussing acid-base chemistry (ie, HA + H₂O → H₃O⁺ + A^–; B^– + H₂O → HB + OH^–). However, this is not a defining property of all acids or bases.

The Lewis classification offers the least-restrictive definition of such substances. A Lewis acid is an electron acceptor and a Lewis base is an electron donor.

Simplistically, acids are sour and turn litmus paper red; bases feel slippery (due to the dissolution of skin), are bitter, and turn litmus paper blue. Of note, the tasting and tactile assessment of substances to determine their acid-base status is not recommended.

Because acidity and alkalinity are determined by the number of available H⁺ ions, it is useful to classify chemicals by their effect on the H⁺ concentration. Strong acids ionize almost completely in aqueous solution, and very little of the parent compound remains. Thus, 0.001 (or 10^–3) mol of HCl, a strong acid, added to 1 L of water produces a solution with a pH of 3. Weak acids, on the other hand, reach an equilibrium between parent and ionized forms, and thus do not alter the pH to the same degree as a similar quantity of a strong acid. This chemical notation defines the strength or weakness of an acid and should not be confused with the concentration of the acid. Thus, the pH of a dilute strong acid solution is substantially less than that of a concentrated weak acid (Table 10–2).

The degree of ionization of an acid is determined by the pK_a, or the negative log of the ionization constant, which represents the pH at which an acid is half dissociated in solution. The same relationship applies to the pK_b of an alkali, although pK_b is rarely used and the degree of ionization of bases is also expressed as pK_a (of note, pK_a = 14 – pK_b). The lower the pK_a, the stronger the acid; the higher the pK_a, the stronger the base. The pK of a strong acid is clinically irrelevant because it is nearly fully ionized under all but the most extremely acidic conditions. Importantly, knowledge of the pK_a does not itself denote the strength of an acid or an alkali. To some extent, this quality is predicted by its chemical structure or reactivity, or it can be obtained through direct measurement or from a reference source.

Because only uncharged compounds cross lipid membranes spontaneously, the pK_a has strong clinical relevance. Salicylic acid, a weak acid with a pK_a of 3, is nonionized in the stomach (pH = 2) and passive absorption occurs (Fig. 9–3). Because it is predominantly in the ionized form (ie, salicylate) in blood, which has a pH of 7.4, little of the ionized blood-borne salicylate passively enters the tissues. However, because in overdose the serum salicylate rises considerably, enough enters the tissue to result in clinical toxicity. Salicylate is the conjugate base of a weak acid (and thus itself a strong base). It diffuses into the mitochondrial intermembrane space (between the inner and outer mitochondrial membrane), where abundant protons exist that have been transported there via the electron transport chain of this organelle (Fig. 11–3). Because salicylate is a strong base, it protonates easily in this environment. In this nonionized form, some of the salicylic acid passes through the inner mitochondrial membrane, into the mitochondrial matrix, and again establish equilibrium by losing a proton. This is the uncoupling of oxidative phosphorylation, dispersing highly concentrated protons in the intermembrane space that are normally used to generate adenosine triphosphate (Chap. 11). Uncoupling contributes to a metabolic acidosis, which shifts the blood equilibrium of aspirin toward the nonionized, protonated form (salicylic acid), enabling it to cross the blood-brain barrier. Presumably, once in the brain, the salicylate uncouples the metabolic activity of neurons with the subsequent development of cerebral edema. Part of the treatment of patients with salicylate toxicity is interrupting the above chain of events with serum alkalinization (Chap. 37).

In a related manner, alkalinization of the patient’s urine prevents reabsorption by ionization of the urinary salicylate. Salicylate anion excreted into acidic urine protonates, and the uncharged moiety then back-diffuses across the renal tubular epithelium and re-enter the body. Alkalinization of the urine “traps” salicylate in the deprotonated form, enhancing excretion. Conversely, because cyclic antidepressants are organic bases, alkalinization of the urine reduces their ionization and actually decreases the urinary elimination of the drug. However, in the management of cyclic antidepressant poisoning, because the other beneficial effects of sodium bicarbonate on the sodium channel outweigh the negative effect on drug elimination, serum alkalinization is recommended (Chap. 68).

ORGANIC CHEMISTRY

The study of carbon-based chemistry and the interaction of inorganic molecules with carbon-containing compounds is called organic chemistry, because the chemistry of living organisms is carbon based. Biochemistry (Chap. 11) is a subdivision of organic chemistry; it is the study of organic chemistry within biologic systems. This section reviews many of the salient points of organic chemistry, focusing on those with the most applicability to medicine and the study of toxicology: nomenclature, bonding, nucleophiles and electrophiles, stereochemistry, and functional groups.

Chemical Properties of Carbon

Carbon, atomic number 6, has a molecular weight of 12.011 g/mol. With few exceptions (notably cyanide ion and carbon monoxide), carbon forms 4 bonds in stable organic molecules. In organic compounds, carbon is commonly bonded to other carbon atoms, as well as to hydrogen, oxygen, nitrogen, or halides. Under certain circumstances, carbon is bonded to metals, as is the case with methylmercury.

Nomenclature

The most systematic method to name organic compounds is in accordance with standards adopted by the International Union of Pure and Applied Chemistry (IUPAC); these names are infrequently used, especially for larger molecules, and alternative names are common. The complete details of the IUPAC naming system are beyond the scope of this text and can be reviewed elsewhere (www.iupac.org), but a brief description of the fundamentals of this system is included here.

The carbon backbone of a molecule serves as the basis of its chemical name. Once the carbon backbone is identified and named, substituents (atoms or groups of atoms that substitute for hydrogen atoms) are identified, named, and numbered. The number refers to the carbon to which the substituent is attached. Some of the common substituents in organic chemistry are –OH (hydroxy), –NH₂ (amino), –Br (bromo), –Cl (chloro), and –F (fluoro). Substituents are then alphabetized and placed as prefixes to the carbon chain.

As an example, consider the molecule 2-bromo-2-chloro-1,1,1-trifluoroethane. The molecule has a 2-carbon backbone (ethane), 3 fluoride atoms on the first carbon, a bromine atom on the second carbon, and a chlorine atom on the second carbon (Fig. 10–4A). A basic understanding of a few simple rules of nomenclature thus allows one to quickly generate the molecular structure of a familiar compound, halothane.

Although the above-mentioned rules suffice to name simple structures, they are inadequate to describe many others, such as molecules with complex branching or ring structures. The IUPAC rules for naming compounds such as [1R-(exo,exo)]-3-(benzoyloxy)-8-methyl-8-azabicyclo[3,2,1]octane-2-carboxylic acid methyl ester, for example, are too complex to include here. Fortunately, many compounds with complex chemical names have simpler names for day-to-day use; as an example, this molecule is commonly referred to as cocaine (Fig. 10–4B).

Cocaine is an example of a common or trivial name: one without a systematic basis, but which is generally accepted as an alternative to (frequently unwieldy) proper chemical names. Common names often refer to the origin of the substance; for example, cocaine is derived from the coca leaf, and wood alcohol (methanol) can be prepared from wood. Alternatively, a common name frequently refers to the way in which a compound is used; for example, rubbing alcohol is a common name for isopropanol. Common names are often imprecise and generate some confusion, however, as evidenced by the fact that rubbing alcohol, when commercially marketed, is either ethanol or isopropanol.

An even less precise system of nomenclature is the use of street names. A street name is a slang term for a drug of abuse, such as “blow” (cocaine), “weed” (marijuana), or “smack” (heroin). The street name ecstasy refers to the stimulant 3,4-methylenedioxymethamphetamine (MDMA), which is most frequently consumed in pill form. It would stand to reason that liquid ecstasy might refer to a solution of MDMA, but street names are not necessarily logical. Instead, liquid ecstasy refers to the drug γ-hydroxybutyrate, a sedative-hypnotic with a completely different pharmacologic and toxicologic profile. Furthermore, there are no standards for the content of ecstasy, and many street pills contain other chemicals.

A final consideration must be given to product names. Product names are trade names under which a given compound might be marketed and are frequently different from both the chemical name and common name. Thus, the inhalational anesthetic in Fig. 10–4A with the chemical name 2-bromo-2-chloro-1,1,1-trifluoroethane has the common name halothane and the trade name Fluothane.

Bonding in Organic Chemistry

While much of the bonding in inorganic chemistry is ionic, the vast majority of bonding in organic molecules is covalent. Whereas electrons in ionic bonds are described as predominately “belonging” to one atom or another, electrons in covalent bonds are shared between 2 atoms; this type of bonding occurs when the difference in electronegativity between 2 atoms is insufficient for one atom to wrest control of an electron from another. Bonds are represented by lines between the atoms: one for a single bond, 2 for a double bond, and 3 for a triple bond.

Nucleophiles and Electrophiles

Many organic reactions of toxicologic importance are described as the reactions of nucleophiles with electrophiles. Nucleophiles (literally, nucleus-loving) are species with increased electron density, frequently in the form of a lone pair of electrons (as in the cases of cyanide ion and carbon monoxide). Nucleophiles, by virtue of their increased electron density, have an affinity for atoms or molecules that are electron deficient; such moieties are called electrophiles (literally, electron-loving). The electron deficiency of electrophiles is described as absolute or relative. Absolute electron deficiency occurs when an electrophile is charged, as is the case with cations such as Pb²⁺ and Hg²⁺. Relative electron deficiency occurs when one atom or group of atoms shifts electrons away from a second atom, making the second atom relatively electron deficient. This is the case for the neurotoxin 2,5-hexanedione (Fig. 10–5); the electronegative oxygen of the carbon-oxygen double bond pulls electron density away from the second and fifth carbon atoms of this molecule, making these carbon atoms electrophilic.

The reaction of a nucleophile with an electrophile involves the movement of electrons, by forming or breaking bonds. This movement of electrons is frequently denoted by the use of curved arrows, which better demonstrates how the nucleophile and electrophile interact. The interaction of acetylcholinesterase with acetylcholine, organic phosphorus compounds, and pralidoxime hydrochloride provides an example of how the use of curved arrow notation leads to a better understanding of the reactions involved.

Under normal circumstances, the action of acetylcholine is terminated when the serine residue in the active site of acetylcholinesterase attacks this neurotransmitter, forming a transient serine-acetyl complex and liberating choline. This serine-acetyl complex is then rapidly hydrolyzed, producing an acetic acid molecule and regenerating the serine residue for another round of the reaction (Fig. 10–6A). In the presence of an organic phosphorus compound, however, this serine residue attacks the electrophilic phosphate atom, forming a stable serine-phosphate bond, which is not hydrolyzed (Fig. 10–6B). The enzyme, thus inactivated, can no longer break down acetylcholine, leading to an increase of this neurotransmitter in the synapse and possibly a cholinergic crisis.

The enzyme can be reactivated, however, by the use of another nucleophile. Pralidoxime hydrochloride (2-PAM) is referred to as a site-directed nucleophile. Because part of its chemical structure (the charged nitrogen atom) is similar to the choline portion of acetylcholine, this antidote is directed to the active site of acetylcholinesterase. Once in position, the nucleophilic oxime moiety (–NOH) that is remote from the positive charge of pralidoxime attacks the electrophilic phosphate moiety. This displaces the serine residue, regenerating the enzyme (Fig. 10–6C) (Chap. 110 and Antidotes in Depth: A36).

A second toxicologically important electrophile is NAPQI (Fig. 10–7). NAPQI is formed when the endogenous detoxification pathways of APAP metabolism (glucuronidation and sulfation) are overwhelmed (Chap. 33). As a result of the electron configuration of NAPQI, the carbon atoms adjacent to the carbonyl carbon (a carbon that is double bonded to an oxygen) are very electrophilic; the sulfur groups of cysteine residues of hepatocyte proteins react with NAPQI to form a characteristic adduct (formed when one compound is added to another), 3-(cystein-S-yl)APAP, in a multistep process. These adducts are released as hepatocytes die, and can be found in the blood of patients with APAP-related liver toxicity. Figure 10–7 diagrams the mechanism of the protein-NAPQI reaction (Chap. 33).

Nucleophiles are often described by their strength, which by convention is related to the rate at which they react with a reference electrophile (CH₃I). Of more common use in pharmacology and toxicology, however, are the descriptive terms “hard” and “soft.” Although imprecise, the designations hard and soft help to predict, qualitatively, how nucleophiles and electrophiles interact with one another.

Hard species have a charge (or partial charge) that is highly localized; that is, their charge-to-radius ratio is high. Hard nucleophiles are molecules in which the electron density or lone pair is tightly held; fluoride, a small atom that cannot spread its electron density over a large area, is an example. Similarly, hard electrophiles are species in which the positive charge cannot be spread over a large area; ionized calcium, a small ion, is a hard electrophile.

Soft species, on the other hand, are capable of delocalizing their charge over a larger area. In this case the charge-to-mass ratio is low, either because the atom is large or because the charge can be spread over a number of atoms within a given molecule. Sulfur is the prototypical example of a soft nucleophile, and the lead ion, Pb²⁺, is a typical soft electrophile.

The utility of this classification lies in the observation that hard nucleophiles tend to react with hard electrophiles, and soft nucleophiles with soft electrophiles. For example, a principal toxicity of fluoride ion poisoning (Chap. 104) is hypocalcemia; this is because the fluoride ions (hard nucleophiles) readily react with calcium ions (hard electrophiles). On the other hand, the soft nucleophile lead is effectively chelated by soft electrophiles such as the sulfur atoms in the chelators dimercaprol (Antidotes in Depth: A28) and succimer (Antidotes in Depth: A29).¹

Isomerism

Isomerism describes the different ways in which molecules with the same chemical formula (ie, the same number and types of atoms) can be arranged to form different compounds. These different compounds are called isomers. Isomers always have the same chemical formula but differ either in the way that atoms are bonded to each other (constitutional isomers) or in the spatial arrangement of these atoms (geometric isomers or stereoisomers).

Constitutional isomers are conceptually the easiest to understand, because a quick glance shows them to be very different molecules. The chemical formula C₂H₆O, for example, can refer to either dimethyl ether or ethanol (Fig. 10–8). These molecules have very different physical and chemical characteristics, and they have little in common other than the number and type of their atomic constituents.

Stereoisomerism, also referred to as geometric isomerism, refers to the different ways in which atoms of a given molecule, with the same number and types of bonds, might be arranged. The most important type of stereoisomerism in pharmacology and toxicology is the stereochemistry around a stereogenic (sometimes called chiral) carbon.

Consider the 2 representations of halothane shown in Fig. 10–9. In this figure, the straight solid lines and the atoms to which they are bonded exist in the plane of the paper, the solid triangle and the atom to which it is bonded are coming out of the paper, and the dashed triangle and the atom to which it is bonded are receding into the paper. It is clear that, for the molecules in Figs. 10–9A and 10-9B, no amount of rotation or manipulation will make these molecules superimposable. They are, therefore, different compounds.

These molecules are enantiomers or optical isomers. They differ only in the way in which their atoms are bonded to a chiral carbon.⁶ It is important to define the stereochemical configuration of these 2 molecules, which is done in one of 2 ways. In the first classification—the d(+)/l(–) system—molecules are named empirically based on the direction in which they rotate plane-polarized light. Each enantiomer will rotate plane-polarized light in one direction; the enantiomer that rotates light clockwise (to the right) is referred to as d(+), or dextrorotatory; the l(–), or levorotatory, enantiomer rotates plane-polarized light in a counterclockwise fashion (to the left).

Alternatively, enantiomers are named using an elaborate and formal set of rules known as Cahn-Ingold-Prelog. These rules establish priority for substituents, based primarily on molecular weight, and then use the arrangement of substituents to assign a configuration. To correctly assign configuration in this system, the molecule is rotated into a projection in which the chiral carbon is in the plane of the page, the lowest priority substituent is directly behind the chiral carbon (and therefore behind the plane of the page), and the other 3 substituents are arranged around the chiral carbon. Figure 10–10 assigns Cahn-Ingold-Prelog priority to the halothane enantiomers of Fig. 10–9, and rearranges the molecules in the appropriate projections.

If the priority of the substituents increases as one moves clockwise (to the right), the enantiomer is R (Latin, rectus = right); if it increases as one moves counterclockwise, the enantiomer is S (Latin, sinister = left). Thus, the one in Fig. 10–10A is the R enantiomer of halothane and Fig. 10–10B represents the S enantiomer.

Enantiomers have identical physical properties, such as boiling point, melting point, and solubility in different solvents; they differ from each other in only 2 significant ways. The first, as mentioned before, is that enantiomers rotate plane-polarized light in opposite directions; this point has no practical toxicologic importance. The second is that enantiomers interact in different ways with other chiral structures (such as proteins, DNA, and other cell receptors), which is of both pharmacologic and toxicologic significance.⁶

The best analogy to explain the toxicologic and pharmacologic importance of stereochemistry is that of the way a hand (analogous to a molecule of xenobiotic) fits into a glove (analogous to the biologic site of activity). Consider the left hand as the S enantiomer and the right hand as the R enantiomer. There are, qualitatively, 3 different ways in which the hand can fit into (interact with) a glove.

First, if the glove is pliable and relatively fluid (such as a disposable latex glove), it can accept either the left hand or the right hand without difficulty; this is the case for halothane, whose R and S enantiomers interact with cell membranes rather than specific receptors and possess equal activity. Second, if a glove is constructed with greater (but imperfect) specificity, one hand will fit well and the other poorly; this is the case for many substances, such as epinephrine and norepinephrine, whose naturally occurring levorotatory enantiomers are 10-fold more potent than the synthetic dextrorotatory enantiomers. Finally, a glove can be made with exquisite precision, such that one hand fits perfectly, while the other hand does not fit at all. This is the case for physostigmine, in which the (–) enantiomer is biologically active whereas the (+) enantiomer is not.

Even the above analogy is oversimplified, however, as there are examples where one enantiomer of a drug is an agonist whereas the other enantiomer is an antagonist. Dobutamine, has one stereogenic carbon and thus 2 stereoisomers; at the α₁-adrenergic receptor, l-dobutamine is a potent agonist and d-dobutamine is a potent antagonist. Because dobutamine is marketed as a racemic mixture (a racemic mixture is a 1:1 mixture of enantiomers), however, these effects cancel each other out. Interestingly, at the β₁-adrenergic receptor, d- and l-dobutamine have unequal agonist effects, with d-dobutamine approximately 10 times more potent than l-dobutamine.

Functional Groups

There is perhaps no concept in organic chemistry as powerful as that of the functional group. Functional groups are atoms or groups of atoms that confer similar reactivity on a molecule; of less importance is the molecule to which it is attached. Alcohols, carboxylic acids, and thiols are functional groups; hydrocarbons are generally not considered functional groups per se, but rather are the structural backbone to which functional groups are attached. The functional groups discussed below are included to illustrate important principles, not because they represent an exhaustive list of important functional groups in toxicology.

Hydrocarbons, as their name implies, consist of only carbon and hydrogen. Alkanes are hydrocarbons that contain no multiple bonds; they are straight chain, usually designated by the prefix n- as in n-butane (Fig. 10–11A) or iso- if branched, as in isobutane (Fig. 10–11B). Alkenes contain carbon-carbon double bonds. Alkynes, which contain carbon-carbon triple bonds, are of limited toxicologic importance. Butane (found in lighter fluid) is an alkane, and gasoline is a mixture of alkanes.

Hydrocarbons are of toxicologic importance for 2 reasons. They are widely abused as inhalational drugs for their central nervous system (CNS) depressant effects, and cause profound toxicity when aspirated. Although these effects are physiologically disparate, they are readily understood in the context of the chemical characteristics of the hydrocarbon functional group.

Hydrocarbons do not contain polar groups (ie, groups that introduce full or partial charges into the molecule). As such, they interact readily with other nonpolar substances, such as lipids or lipophilic substances. Hydrocarbons readily interact with the myelin of the CNS, disrupting normal ion channel function and causing CNS depression. When aspirated, hydrocarbons interact with the fatty acid tail of surfactant, dissolving this protective substance and contributing to acute respiratory distress syndrome (Chap. 105).

Alcohols possess the hydroxyl (–OH) functional group, which adds polarity to the molecule and makes alcohols highly soluble in other polar substances, such as water. For example, ethane gas (CH₃CH₃) has negligible solubility in water, whereas ethanol (CH₃CH₂OH) is miscible, or infinitely soluble, in water. In biologic systems, alcohols are generally CNS depressants, but they can also act as nucleophiles. Ethanol reacts with cocaine to form cocaethylene, a longer-acting and more vasoactive substance than cocaine itself (Fig. 10–12) (Chap. 75).

Alcohols are categorized as primary, secondary, or tertiary, in which the reference carbon is bonded to 1, 2, or 3 carbons (respectively) in addition to the hydroxyl group. Methanol, in which the reference carbon is bonded to no other carbons, is not a primary alcohol per se but shares many of the reactivity patterns of primary alcohols. The difference between primary, secondary, and tertiary structures is important, because although the alcohol functional group imparts many qualities to the molecule, the degree of substitution can affect the chemical reactivity. Primary alcohols can undergo multistep oxidation to form carboxylic acids, whereas secondary alcohols generally undergo one-step metabolism to form ketones, and tertiary alcohols do not readily undergo oxidation. This is a point of significant toxicologic importance, and is discussed in more detail below.

Alcohols are named in several ways; the most common is to add -ol or -yl alcohol to the appropriate prefix. If the alcohol group is bonded to an interior carbon, the number to which the carbon is bonded precedes the suffix.

Carboxylic acids contain the functional group –COOH. As their name implies, they are (weakly) acidic, and the pK_a of carboxylic acids is generally 4 or 5, depending on the substitution of the molecule. Small-molecular-weight carboxylic acids are capable of producing an elevated anion gap metabolic acidosis, which is true whether the acids are endogenous or exogenous. Examples of endogenous acids are β-hydroxybutyric acid and lactic acid; examples of exogenous acids are formic acid (produced by the metabolism of methanol) and glycolic, glyoxylic, and oxalic acids (produced by the metabolism of ethylene glycol). Carboxylic acids are named by adding -oic acid to the appropriate prefix; the 4-carbon straight-chain carboxylic acid is thus butanoic acid.

Thiols contain a sulfur atom, which usually functions as a nucleophile. The sulfur atom of N-acetylcysteine regenerates glutathione reductase and also reacts directly with NAPQI to detoxify this electrophile. The sulfur atoms of many chelators, such as dimercaprol and succimer, are nucleophiles that are very effective at chelating electrophiles such as heavy metals. Thiols are generally named by adding the word thiol to the appropriate base. Thus, a 2-carbon thiol is ethane thiol.

As noted above, molecules with a given functional group often have more in common with molecules with the same functional group than they have in common with the molecules from which they were derived. The alkanes methane, ethane, and propane are straight-chain hydrocarbons with similar properties. All are gases at room temperature, have almost no solubility in water, and have similar melting and boiling points. When these molecules are substituted with one or more hydroxide functional groups, they become alcohols: examples are methanol, ethanol, ethylene glycol (a glycol is a molecule that contains 2 alcohol functional groups), the primary alcohol 1-propanol, and the secondary alcohol 2-propanol (isopropanol). Each of these alcohols is a liquid at room temperature, and all are very soluble in water. All have boiling points that are markedly different from the alkane from which they were derived, and are very close to each other.

In addition to conferring different physical properties on the molecule, the addition of the alcohol functional group also confers different chemical properties and reactivities. For example, methane, ethane, and propane are virtually incapable of undergoing oxidation in biologic systems. The alcohols formed by the addition of one or more hydroxyl groups, however, are readily oxidized by alcohol dehydrogenase (Fig. 10–13).

As Fig. 10–13 indicates, the oxidation of the primary alcohols methanol and ethanol results in the formation of aldehydes (functional groups in which a carbon atom contains a double bond to oxygen and a single bond to hydrogen), whereas the oxidation of the secondary alcohol isopropanol results in the formation of a ketone (a functional group in which a carbon is double-bonded to an oxygen atom and single-bonded to 2 separate carbon atoms). Although both aldehydes and ketones contain the carbonyl group (a carbon-oxygen double bond), aldehydes and ketones are distinctly different functional groups, and they have different reactivity patterns. For instance, aldehydes can undergo enzymatic oxidation to carboxylic acids (Figs. 10–13A and 13B), whereas ketones cannot (Fig. 10–13C).

It is here that recognition of functional groups helps to understand the potential toxicity of an alcohol. Methanol, ethanol, and isopropanol are all alcohols; as such, their toxicity before metabolism is expected to be (and in fact is) quite similar to that of ethanol, producing CNS sedation. Because these toxins are primary and secondary alcohols, all can be metabolized to a carbonyl compound, either an aldehyde or a ketone. Here, however, the functional groups on the molecules have changed; whereas aldehydes can undergo metabolism to carboxylic acids (which can, in turn, cause an anion gap acidosis), ketones cannot. It is for this reason that methanol and ethylene glycol cause an anion gap acidosis whereas isopropanol does not (Chap. 106).

The concept of functional groups, however useful, has limitations. For example, although both formic acid and oxalic acid are organic acids, they cause different patterns of organ system toxicity. Formic acid is a mitochondrial toxin and exerts effects primarily in areas (such as the retina or basal ganglia) that poorly tolerate an interruption in the energy supplied by oxidative phosphorylation. Conversely, oxalic acid readily precipitates calcium and is toxic to renal tubular cells, which accounts for the nephrotoxicity that is characteristic of severe ethylene glycol poisoning. The concept of the functional group is thus an aid to understanding chemical reactivity, but not a substitute for a working knowledge of the toxicokinetic or toxicodynamic effects of xenobiotics in living systems.

SUMMARY

• Understanding key principles of inorganic and organic chemistry provide insight into the mechanisms by which xenobiotics function.

• The periodic table forms the basis for understanding inorganic chemistry and provides insight into the expected reactivity, and to a large extent the clinical effects, of an element.

• A growing understanding of how reactive species are formed and how they interfere with physiologic processes has led to new insights in the pathogenesis and treatment of toxin-mediated diseases.

• Changes in functional groups on organic molecules dramatically changes their chemical and toxicologic effects, and knowing the relevant general principles is essential to an understanding of biochemistry and pharmacology.

REFERENCES

INTRODUCTION

Xenobiotics are compounds that are foreign to a living system. Toxic xenobiotics interfere with critical metabolic processes, cause structural damage to cells, or alter cellular genetic material. The specific biochemical sites of actions that disrupt metabolic processes are well characterized for many xenobiotics although mechanisms of cellular injury are not. This chapter reviews the biochemical principles that are relevant to an understanding of the biotransformation enzymes and their clinical implications and the damaging effects of toxic xenobiotics.

XENOBIOTIC CHARACTERISTICS AND TOXICITY

The capacity of a xenobiotic to produce injury is affected by many factors, including its absorption, distribution, elimination, genotypic/phenotypic states, synergistic/antagonistic coingestants, site of activation or detoxification, and site of action. This section focuses on how the route of exposure (absorption) and the ability to cross membranes in order to access particular organs (distribution) affect toxicity.

The route of exposure to a xenobiotic often confines damage primarily to one organ, for example, pulmonary injury that follows inhalation of an irritant gas or gastrointestinal (GI) injury that follows ingestion of a caustic. However, most xenobiotics have lipophilic properties that facilitate absorption across cell membranes of organs that are portals of entry into the body: the skin, GI tract, and lungs. Systemically circulating xenobiotics, such as cyanide or carbon monoxide, affect toxicity in multiple organs. The liver is particularly susceptible to xenobiotic-induced injury as it receives blood supply from both the portal venous system and from the hepatic artery containing xenobiotics absorbed from multiple sites of exposure.

Once systemically absorbed, various factors affect the distribution of a xenobiotic to a particular organ, including its molecular weight, protein binding, plasma pH/drug pKa, lipophilicity, in addition to the presence of membrane transporters, and physiologic barriers (Chap. 9). Restriction of distribution or preferential distribution into a target organ determines the ability of a xenobiotic to mediate tissue injury. For example, many potentially toxic xenobiotics fail to produce central nervous system (CNS) injury because they cannot cross the blood–brain barrier. The negligible CNS effects of the mercuric salts when compared with organic mercury compounds are related to their relative inability to penetrate the CNS. With respect to target organs, 2 potent biologic xenobiotics—ricin (from Ricinus communis) and α-amanitin (from Amanita phalloides)—block protein synthesis through the inhibition of RNA polymerase. However, they result in different clinical effects because they access different tissues. Ricin has a special binding protein that enables it to gain access to the endoplasmic reticulum in GI mucosal cells, where it inhibits cellular protein synthesis and causes severe diarrhea. α-Amanitin is transported into hepatocytes by bile salt transport systems, where inhibition of protein synthesis results in cell death. Finally, the electrical charge of a xenobiotic at tissue pH affects its ability to enter a cell. Unlike the ionized (charged) form of a xenobiotic, the un-ionized (uncharged) form is often lipophilic and passes through lipid cell membranes to enter the cells. The pK_a of an acidic xenobiotic (HA⇌A^– + H⁺) is the pH at which 50% of the molecules are ionized (A^– form) and 50% is un-ionized (HA form). A xenobiotic with a low pK_a is more likely to be absorbed in an acidic environment in which the un-ionized form predominates. This conept is the basis for urine alkalinization and ion trapping in the management of salicylate toxicity (Chap. 37).

GENERAL ENZYME CONCEPTS

The capability to detoxify and eliminate both endogenous toxins and xenobiotics is crucial to the maintenance of physiologic homeostasis. A simple example is the detoxification of cyanide, a potent cellular poison that is common in the environment and is also a product of normal metabolism. Mammals have evolved to have the enzyme rhodanese, which combines cyanide with thiosulfate to create the less toxic, renally excreted compound thiocyanate.⁷

The liver has the highest concentration of enzymes that metabolize xenobiotics. Enzymes found in the cytosol of hepatocytes that are specific for alcohols, aldehydes, esters, or amines act on many different substrates within these broad chemical classes. Enzymes that act on more lipophilic xenobiotics, including the cytochrome enzymes, are embedded in the lipid membranes of the cytosol-based endoplasmic reticulum. Cytochromes are a class of hemoprotein enzymes whose function is electron transfer, using a cyclical transfer of electrons between oxidized (Fe³⁺) or reduced (Fe²⁺) forms of iron. One type of cytochrome is cytochrome P450 (CYP) whose nomenclature derives from the spectrophotometric characteristics of its associated heme molecule. When bound to carbon monoxide, the maximal absorption spectrum of the reduced CYP (Fe²⁺) enzyme occurs at 450 nm.⁷⁹ These CYP enzymes commonly split the 2 oxygen atoms of an oxygen molecule, incorporating one into the substrate and one into water and thus are called mixed-function oxidases or monooxygenases (Fig. 11–1). This differs from dioxygenases that incorporate both oxygen atoms into the substrate.^79,108

Microsomes are the pieces of the endoplasmic reticulum that result when cells are disrupted. When cells are mechanically disrupted and centrifuged, these membrane-bound enzymes are found in the pellet, or microsomal fraction; hence, they are called microsomal enzymes. Enzymes located in the liquid matrix of cells are called cytosolic enzymes and are found in the supernatant when disrupted cells are centrifuged.²²

BIOTRANSFORMATION OVERVIEW

The study of xenobiotic metabolism was established as a scientific discipline by the seminal publication of Williams in 1949.¹²⁰ Biotransformation is the physiochemical alteration of a xenobiotic, usually as a result of enzyme action. Most definitions also include that this action converts lipophilic substances into more polar, excretable substances.^70,108 Most xenobiotics undergo some biotransformation, the degree of which is affected by their chemical nature. The hydrophilic nature of ionized compounds such as carboxylic acids enables the kidneys to rapidly eliminate them. Very volatile compounds, such as enflurane, are expelled promptly via the lungs. Neither of these groups of xenobiotics undergo significant enzymatic metabolism.

Biotransformation usually results in “detoxification,” a reduction in the toxicity, by the conversion to hydrophilic metabolites of the xenobiotic that can be renally eliminated.⁷⁰ However, this is not always the case. Many parent xenobiotics are inactive and must undergo “metabolic activation.”⁷² When metabolites are more toxic than the parent xenobiotic, biotransformation is said to have resulted in “toxification.”¹⁰⁸ Biotransformation via acetylation or methylation enhances the lipophilicity of a xenobiotic. Biotransformation is accomplished by impressively few enzymes, reflecting broad substrate specificity. The predominant pathway for the biotransformation of an individual xenobiotic is determined by many factors, including the availability of cofactors, changes in the concentration of the enzyme caused by induction, and the presence of inhibitors. The predominant pathway is also affected by the rate of substrate metabolism, reflected by the K_m (Michaelis–Menten dissociation constant) of the biotransformation enzyme (Chap. 9).¹⁰⁸

Biotransformation is often divided into phase I and phase II reactions, terminology first introduced in 1959.¹²⁰ Phase I reactions prepare lipophilic xenobiotics for the addition of functional groups or actually add the groups, converting them into more chemically reactive metabolites. This is usually followed by phase II synthetic reactions that conjugate the reactive products of phase I with other molecules that render them more water soluble, further detoxifying the xenobiotics and facilitating renal elimination. However, biotransformation often does not follow this stepwise process.⁵² Some xenobiotics undergo only a phase I or a phase II reaction prior to elimination. Additionally, phase II reactions can precede phase I. While virtually all phase II synthesis reactions cause inactivation, a classic exception is fluoroacetate being metabolized to fluorocitrate, a potent inhibitor of the citric acid (tricarboxylic acid, or Krebs) cycle (Chap. 113).⁹⁰

Biotransformed xenobiotics cannot be eliminated until they are moved back across cell membranes and out of the cells. Membrane transporters are proteins that move endobiotics/xenobiotics across the membranes without altering their chemical compositions Most membrane transporters are in the adenosine triphosphate binding cassette (ABC) family of transmembrane proteins that use energy from adenosine triphosphate (ATP) hydrolysis to move xenobiotics.^14,45,52 This family includes the P-glycoprotein family and the glutathione S-conjugate export pump.¹⁰³ The process of transport is sometimes termed a “phase III reaction.” However, membrane transport does not always occur after phase I or II reactions, and some parent compounds, such as digoxin, are transported across membranes without any biotransformation at all.²⁵

Phase I Biotransformation Reactions

Oxidation is the predominant phase I reaction, adding reactive functional groups suitable for synthetic conjugation during phase II. These groups include hydroxyl (–OH), sulfhydryl (–SH), amino (–NH₂), aldehyde (–CHO), or carboxyl (–COOH) moieties. Noncarbon elements such as nitrogen, sulfur, and phosphorus are also oxidized in phase I reactions. Other phase I reactions include hydrolysis (the splitting of a large molecule by the addition of water that is divided among the 2 products), hydration (incorporation of water into a complex molecule), hydroxylation (the attachment of –OH groups to carbon atoms), reduction, dehalogenation, dehydrogenation, and dealkylation.^70,108

The CYP enzymes are the most numerous and important of the phase I enzymes. A common oxidation reaction catalyzed by CYP enzymes is illustrated by the hydroxylation of a xenobiotic R–H to R–OH (Fig. 11–1).³⁰ Membrane-bound flavin monooxygenase (FMO), a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase located in the endoplasmic reticulum, is an important oxidizer of amines and other compounds containing nitrogen, sulfur, or phosphorus.⁷⁰

The alcohol, aldehyde, and ketone oxidation systems use predominantly cytosolic enzymes that catalyze these reactions using nicotinamide adenine dinucleotide (NADH/NAD⁺), but alcohol oxidation can also occur in microsomes via CYP enzymes and in peroxisomes via the enzyme catalase (Fig. 11–2).^65,108,123 Two classic phase I oxidation reactions are the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase (ADH) followed by the metabolism of acetaldehyde to acetic acid by aldehyde dehydrogenase (ALDH). Alcohol dehydrogenase, which oxidizes many different alcohols, is found in the liver, lungs, kidneys, and gastric mucosa.⁶⁵ Variations in these enzymes have clinical implications. For example, young women have less ADH in their gastric mucosa than young men. This results in decreased first-pass metabolism of ethanol and increased ethanol absorption. Some populations, particularly Asians, are deficient in ALDH, resulting in increased acetaldehyde concentrations and symptoms (Chap. 78).⁶⁵

Oxidation Overview

Biotransformation often results in the oxidation or reduction of carbon. A substrate is oxidized when it transfers electrons to an electron-seeking (electrophilic or oxidizing) molecule, leading to reduction of the electrophilic molecule. These oxidation-reduction reactions are usually coupled to the cyclical oxidation and reduction of a cofactor, such as the pyridine nucleotides, NADH/NAD⁺, or NADPH/NADP⁺. The nucleotides alternate between their reduced (NADPH, NADH) and oxidized (NADP⁺, NAD⁺) forms. Because xenobiotic oxidation is the most common phase I reaction, the newly created reduced cofactors must have a place to unload their electrons; otherwise, biotransformation ends. The electron transport chain serves as the major electron recipient.

Electrons resulting from the catabolism of energy sources are extracted primarily by NAD⁺, forming NADH. Within the mitochondria, NADH transports its electrons to the cytochrome-mediated electron transport chain. This results in the production of ATP, the reduction of molecular oxygen to water, and the regeneration of NAD⁺—all parts critical to the maintenance of oxidative metabolism. Nicotinamide adenine dinucleotide phosphate, generated by the hexose monophosphate shunt, is used in the synthetic (anabolic) reactions of biosynthesis (especially fatty acid synthesis). Nicotinamide adenine dinucleotide phosphate is also the cofactor in the reduction of glutathione, a molecule vital to the protection of cells from oxidative damage.

Biotransformation often results in the oxidation or reduction of carbon. The oxidation state of a specific carbon atom is determined by counting the number of hydrogen and carbon atoms to which it is connected. The more reduced a carbon, the higher the number of connections. For example, the carbon in methanol (CH₃OH) has 3 carbon–hydrogen bonds and is more reduced than the carbon in formaldehyde (H₂C=O), which has two. Carbon–carbon double bonds count as only one connection.

Cytochrome Enzymes—An Overview

Cytochrome enzymes perform many functions. The biotransformation CYP enzymes are bound to the lipid membranes of the smooth endoplasmic reticulum. They execute 75% of all xenobiotic metabolism and most phase I oxidative biotransformations of xenobiotics.³⁹ A second role for CYP enzymes is synthetic: biotransforming endobiotics (chemicals endogenous to the body) to cholesterol, steroids, bile acids, fatty acids, prostaglandins, and other important lipids. Cytochromes also act within the mitochondrial electron transport chain.^41,78

Although more than 6,000 CYP genes exist in nature, the human genome project determined that the number of human CYP genes was 57.⁷⁸ Cytochrome P450 enzymes are categorized according to the similarities of their amino acid sequences. They are in the same “family” if they are more than 40% comparable and same “subfamily” if they are more than 55% similar.⁷⁷ Families are designated by an Arabic numeral (n), subfamilies by a capital letter (X), and each individual enzyme by another numeral (m), resulting in the nomenclature CYPnXm for each enzyme. For example, CYP3A4 is enzyme number 4 of the CYP3A subfamily of the CYP3 family.⁷⁴ Genes coding for these enzymes contain sequence variations, designated by italicized style and an asterisk followed by an Arabic numeral (ie, CYP2D6*4), that lead to functional differences.^49,74 An allele is a form of a gene that often arises through mutation. If the same gene variation exists in greater than 1% of the human population, then the allele is termed a genetic polymorphism. Most xenobiotic metabolism is done by the CYP1, CYP2, and CYP3 families, with a small amount done by the CYP4 family.^13,117 Although 15 CYP enzymes metabolize xenobiotics,⁸⁶ nearly 90% is done by 6 CYP enzymes: 1A2, 2C9, 2C19, 2D6, 2E1, and 3A4 (Table 11–1).⁷⁸

Most CYP enzymes are found in the liver, where they comprise 2% of the total microsomal protein.⁸⁶ High concentrations are also found in extrahepatic tissues, particularly the GI tract and kidney.^26,80 The lungs,¹²⁶ heart,⁸⁴ and brain²⁷ have the next highest amounts. Each tissue has a unique profile of CYP enzymes that determines its sensitivity to different xenobiotics.²⁶ The CYP enzymes in the enterocytes of the small intestine actually contribute significantly to “first-pass” metabolism of some xenobiotics.^56,78 Corrected for tissue mass, the CYP enzyme system in the kidneys is as active as that in the liver. The activity of the renal CYP enzymes is decreased in patients with chronic kidney disease, with relative sparing of CYPs 1A2, 2C19, and 2D6 compared with 3A4 and 2C9.¹³

Cytochrome P450 Enzyme Specificity for Substrates

In vitro models are used to define the specificities of CYP enzymes for their substrates and inhibitors. However, activity in a test tube does not always correlate with that in a cell. These models use substrate and inhibitor concentrations that are much higher than would be encountered in vivo, and the mathematical models that extrapolate to clinically relevant processes yield conflicting results. Discrepancies in reported substrates, inhibitors, and inducers of specific CYP enzymes results.¹¹⁹

Most CYP enzymes involved in xenobiotic biotransformation have broad substrate specificity and can metabolize many xenobiotics.³⁹ This is fortunate because the number of xenobiotic substrates likely already exceeds 200,000 and continues to grow.⁶⁴ Broad substrate specificity often results in multiple CYP enzymes being able to biotransform the same xenobiotic. This enables the ongoing biotransformation despite an inhibition or deficiency of an enzyme. When a substrate is biotransformed by more than one enzyme, the enzyme with the highest affinity for the substrate usually predominates at low substrate concentrations, whereas enzymes with lower affinity become important at high concentrations. This transition is usually concomitant with, but not dependent on, the saturation of the catalytic capacity of the primary enzyme as it reaches its maximum rate of activity.³⁹ The K_m, which is defined as the concentration of substrate that results in 50% of maximal enzyme activity, describes this property of enzymes. For example, ADH in the liver has a very low K_m for ethanol, making it the primary metabolic enzyme for ethanol when concentrations are low.⁶⁵ Ethanol is also biotransformed by the CYP2E1 enzyme, which has a high K_m for ethanol and only functions when ethanol concentrations are high. The CYP2E1 enzyme metabolizes little ethanol in moderate drinkers but accounts for significantly more biotransformation in alcoholics, as it is also inducible. As another example, diazepam is metabolized by both CYP2C19 and CYP3A4 enzymes. However, the affinity of CYP3A4 for diazepam is so low (ie, the K_m is high) that most diazepam is metabolized by CYP2C19.⁴⁴

The substrate selectivity of some CYP enzymes is determined by molecular and physicochemical properties of the substrates. The CYP1A subfamily has greater specificity for planar polyaromatic substrates such as benzo[a]pyrene. The CYP2E enzyme subfamily targets low-molecular-weight hydrophilic xenobiotics, whereas the CYP3A4 enzyme has increased affinity for lipophilic compounds. Substrates of CYP2C9 are usually weakly acidic, whereas those of CYP2D6 are more basic.⁶⁴ High specificity also results from key structural considerations such as stereoselectivity. Some xenobiotics are racemic mixtures of 2 stereoisomers that are substrates for different CYP enzymes and have distinct affinities for the enzymes, resulting in different rates of metabolism. For example, R-warfarin is biotransformed by CYP3A4 and CYP1A2, whereas S-warfarin is metabolized by CYP2C9.⁹⁷

The CYP enzymes that biotransform a specific xenobiotic cannot be predicted by its drug class. For example, the selective serotonin reuptake inhibitors (SSRIs) fluoxetine and paroxetine are both major substrates and potent inhibitors of CYP2D6, but sertraline is not extensively metabolized by this enzyme and exhibits minimal interaction with other antidepressants.⁵ Most β-hydroxy-β-methylglutarylcoenzyme A (HMG-CoA) reductase inhibitors are metabolized by CYP3A4 (lovastatin, simvastatin, and atorvastatin); however, fluvastatin is metabolized by CYP2D6, and pravastatin undergoes virtually no CYP enzyme metabolism at all.⁶⁹ Among angiotensin-II receptor blockers, losartan and irbesartan are metabolized by CYP2C9, whereas valsartan, eprosartan, and candesartan are not substrates for any CYP enzyme. In addition, losartan is a prodrug whose active metabolite provides most of the pharmacologic activity, whereas irbesartan is the primary active compound. For these 2 drugs, the inhibition of CYP2C9 is predicted to have opposite effects.³¹

Cytochrome P450 and Drug–Drug/Drug–Xenobiotic Interactions

Adverse reactions to medications and drug–drug interactions are common causes of morbidity and mortality, the risk of which increases with the number of drugs taken (Chaps. 134 and 135). As many as 50% of adverse reactions are related to pharmacogenetic factors.³⁴ The most significant interactions are mediated by CYP enzymes.⁷⁸ The impacts of genetic polymorphism and enzyme induction or inhibition are addressed below.

The CYP enzymes are involved in many types of drug interactions. The ability of potential new drugs to induce or inhibit enzymes is an important consideration of industry. Drug development focuses on the potential of new xenobiotics to induce or inhibit other drugs or enzymes during the drug discovery phase. Various in vitro models enable this early determination.⁶⁶

Many xenobiotics interact with the CYP enzymes. St. John’s wort, an herb marketed as a natural antidepressant, induces multiple CYP enzymes, including 1A2, 2C9, and 3A4. The induction of CYP3A4 by St. John’s wort is associated with a 57% decrease in effective serum concentrations of indinavir when given concomitantly.⁸⁸ Xenobiotics contained in grapefruit juice, such as naringin and furanocoumarins, are both substrates and inhibitors of CYP3A4. They inhibit the first-pass metabolism of CYP3A4 substrates by inhibiting CYP3A4 activity in both the GI tract and the liver.²¹ Polycyclic hydrocarbons found in charbroiled meats and in cigarette smoke induce CYP1A2. Thus, for smokers who drink coffee, concentrations of caffeine, a CYP1A2 substrate, will increase following a permanent cessation of smoking.³⁰

Genetic Polymorphism

The response to xenobiotics and to coadministration of inhibitory or inducing xenobiotics is highly variable. The translation of DNA sequences into proteins results in the phenotypic expression of the genes. When a genetic mutation occurs, the changed DNA may continue to exist, be eliminated, or propagate into a polymorphism. A polymorphism is a genetic change that exists in at least 1% of the human population.^34,74 A polymorphism in a biotransformation enzyme may change its rate of activity. The heterogeneity of CYP enzymes contributes to the differences in metabolic activity among patients.³⁴ Differences in biotransformation capacity that lead to toxicity, once thought to be “idiosyncratic” drug reactions, are likely caused by these inherited differences in the genetic complement of individuals.

An individual with 2 CYP alleles of “normal” catalytic speed is called extensive metabolizer, which usually represents a majority of the population. There are 3 major metabolizer phenotypes due to polymorphism: poor, intermediate, and ultrarapid. Poor and intermediate metabolizers have either loss-of-function variants or absent CYP alleles. Poor metabolizers have 2 inactive or absent alleles, whereas intermediate metabolizers will have either 2 reduced function alleles or one “normal” allele with one inactive or absent allele. On the other hand, an ultrarapid metabolizer will have either gain-of-function variants or gene duplication.^34,125 The CYP2C19 and CYP2D6 genes are highly polymorphic (Table 11–1). The CYP2D6 gene, which has 157 alleles, is associated with both ultrarapid and poor metabolism.⁴⁹ The CYP2C19 and CYP2C9 genes are both associated with polymorphisms, resulting in poor metabolizers.^13,78

The clinical implications of polymorphisms are vast. Prodrugs have limited activation in patients who are poor metabolizers. For example, clopidogrel mediates antiplatelet effects via an active thiol metabolite generated by CYP enzymes. Individuals with the loss-of-function CYP2C19*2 allele have decreased generation of the active metabolite, decreased antiplatelet effects, and increased cardiovascular events while on clopidogrel.^55,99 Conversely, some ultrarapid metabolizers generate toxic concentrations of an active metabolic. Codeine (inactive) metabolizes to morphine (active) via CYP2D6. Ultrarapid metabolizers with CYP2D6 can have 2 to 13 copies of the gene leading to rapid conversion of codeine to morphine and as a consequence toxicity.⁶⁷ Finally, some active drugs with inactive metabolites fail to reach therapeutic concentrations in patients who are ultrarapid metabolizers.³⁴

Polymorphisms exist for enzymes other than CYP enzymes. A classic example is the inheritance of rapid or slow “acetylator” phenotypes. Acetylation is important for the biotransformation of amines (R–NH₂) or hydrazines (NH₂–NH₂). Slow acetylators are at increased risk of toxicity associated with the slower biotransformation of certain nitrogen-containing xenobiotics such as isoniazid, procainamide, hydralazine, and sulfonamides.¹⁰¹ Genetic polymorphism of UDP-glucuronosyl transferase 2B17 (UGT2B17) contributes to a bimodal distribution of urinary conjugated testosterone excretion. Since the testosterone isomer, epitestosterone, has little genetic variation and is not affected by testosterone metabolism, an elevated testosterone:epistestosterone (T:E) is indicative of the exogenous administration of testosterone.¹¹⁵ The World Anti-Doping Agency sets the suspicious threshold ratio of T:E ratio at 4:1 in response to genetic variations.^95,121 Athletes with homozygote deletions of UGT2B17 have less conjugation of testosterone and have had false negative testing using the previous ratio of 6:1 (Chap. 41).⁵¹

Polymorphic genes that code for enzymes in important metabolic pathways affect the toxicity of a xenobiotic by altering the response to, or the disposition of, the xenobiotic. An example occurs in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Glucose-6-phosphate dehydrogenase is a critical enzyme in the hexose monophosphate shunt, a metabolic pathway located in the red blood cell (RBC) that produces NADPH, which is required to maintain RBC glutathione in a reduced state. In turn, reduced glutathione prevents hemolysis during oxidative stress.¹⁵ In patients deficient in G6PD, oxidative stress produced by electrophilic xenobiotics results in hemolysis.

Induction of CYP Enzymes

Biotransformation by induced CYP enzymes results in either increased activity of prodrugs or enhanced elimination of drugs. Stopping an inducer results in the opposite effects. Either way, maintaining therapeutic concentrations of affected drugs is difficult, resulting in either toxic or subtherapeutic concentrations. Interestingly, not all CYP enzymes are inducible. The inducible enzymes include CYP1A, CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A.⁶⁶

Although varied mechanisms of induction exist, the most common and significant is nuclear receptor (NR)-mediated increase in gene transcription.⁶⁶ Nuclear receptors are the largest group of transcription factors (proteins) that switch genes on or off.¹¹³ They regulate reproduction, growth, and biotransformation enzymes, including CYP enzymes.¹⁰⁹ Nuclear receptors exist mostly within the cytoplasm of cells. The CYP families 2 and 3 both have gene activation triggered through the NR pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). The CYP1A subfamily uses the aryl hydrocarbon receptor (AhR) as its NR. Ligands, molecules that bind to and affect the reactivity of a central molecule, are typically small and lipophilic, enabling them to enter cells. Many xenobiotics are themselves ligands. Ligands bind the NRs, resulting in structural changes that enable the NR–ligand complexes to be translocated into the cell nucleus. Within the nucleus, NR–ligand complexes bind to proteins to create a heterodimer such as the retinoid X receptor (RXR) with either the PXR or the CAR. Similarly, the AhR interacts with the AhR nuclear translocator (Arnt) to create the nuclear heterodimer. This new heterodimer complex then interacts with specific response elements of DNA, initiating the transcription of a segment of DNA, and translation of its RNA resulting in the phenotypic expression of the respective CYP enzyme.

The ligand-binding domain of the PXR is very hydrophobic and flexible, enabling this pocket to bind many substrates of varied sizes and reflecting why the PXR is activated by a broad group of ligands.^85,109 For example, xenobiotic ligands that bind the NR PXR that targets the CYP3A4 gene include rifampin, omeprazole, carbamazepine, and troleandomycin. Phenobarbital, a classic inducing xenobiotic, is a ligand that binds the CAR.¹¹³ The induction of CYP1A subfamily enzymes is through the interaction with the NR AhR. Exogenous AhR ligands are hydrophobic, cyclic, planar molecules. Classic AhR ligands include polyaromatic hydrocarbons (PAHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene.⁸⁵

Induction requires time to occur because it involves de novo synthesis of new proteins. Similarly, withdrawal of the inducer results in a slow return to the original enzyme concentration.⁶⁶ Polyaromatic hydrocarbons (PAHs) result in CYP1A subfamily induction within 3 to 6 hours with a maximum effect within 24 hours.¹⁰⁴ The inducer rifampin does not affect verapamil trough concentrations maximally until one week; followed by a 2-week return to baseline steady state after withdrawal of rifampin.⁶⁶ Xenobiotics with long half-lives require longer periods of time to reach steady-state concentrations that maximize induction. Phenobarbital and fluoxetine, which have long half-lives, fully manifest induction only after weeks of exposure. Conversely, xenobiotics with short half-lives, such as rifampin or venlafaxine reach maximum induction within days.¹³

Inconsistency in CYP induction exists in all organs and among individuals.^66,85 In an in vitro study of the effects of inducing xenobiotics on 60 livers, differences in enzyme induction ranged from 5-fold for CYP3A4 and CYP2C9 up to more than 50-fold for CYP2A6 and CYP2D6.⁶⁶ The inconsistency likely results from both polymorphisms and multiple environmental factors including diet, tobacco, and pollutants.⁶⁶ There is variation in the extent to which inducers generate new CYP enzymes. Identical dosing regimens with rifampin resulted in induction of in vivo hepatic CYP3A4 with up to 18-fold differences between subjects.⁶⁶ There is an inverse correlation of the degree of inducibility of an enzyme and the baseline enzyme concentration. Patients with a relatively low baseline concentration of a CYP enzyme will be more inducible than those with a high baseline concentration. Interestingly, the maximum concentrations of CYP enzymes seem to be quantitatively similar among individuals, suggesting a limit to which enzymes can be induced.⁶⁶

Although the focus of this section is on CYP enzymes, it appears that most phases of xenobiotic metabolism are regulated by NRs.¹¹ Also, just as genetic polymorphisms exist for CYP enzymes, they exist for NRs including the AhR, the CAR, and the PXR. This results in varied sensitivities to the ligands that complex with the NRs, ultimately resulting in differences in CYP enzyme induction.^66,113

Inhibition of CYP Enzymes

Inhibition of CYP enzymes result in either increased bioavailability of a drug or decreased bioactivation of a prodrug, and is the most common cause of harmful drug–drug interactions.⁸⁶ Severe inhibition of CYP enzymes by a coadministered xenobiotic resulted in the removal of many medications from the market, including terfenadine, mibefradil, bromfenac, astemizole, cisapride, cerivastatin, and nefazodone.¹¹⁹ The appendix at the end of this chapter includes a comprehensive listing of CYP enzyme substrates, inhibitors, and inducers.

Inhibition mechanisms include irreversible (mechanism-based inhibition) and, the more common, reversible processes. The most common type of reversible inhibition is competitive, where the substrate and inhibitor both bind the active site of the enzyme.^86,119 Binding is weak and is formed and broken down easily, resulting in subsequent enzyme liberation. It occurs rapidly, usually beginning within hours.¹³ Because the degree of inhibition varies with the concentration of the inhibitor, the time to reach the maximal effect correlates with the half-life of the xenobiotic in question.¹³ A competitive inhibitor can be overcome by increasing the substrate concentration. Each substrate of a CYP enzyme is an inhibitor of the metabolism of all the other substrates of the same enzyme, thereby increasing their concentrations and half-lives. Reversible, noncompetitive inhibition occurs when an inhibitor binds a location on an enzyme that is not the active site, resulting in a structural change that inhibits the active enzyme site. For example, noncompetitive inhibitors of CYP2C9 include nifedipine, tranylcypromine, and medroxyprogesterone.¹⁰⁰ Another reversible mechanism results from competition between one xenobiotic and a metabolite of a second xenobiotic at its CYP enzyme substrate binding site. For example, the metabolites of clarithromycin and erythromycin produced by CYP3A inhibit further CYP3A activity. The effect is reversible and usually increases with repeated dosing.¹⁰⁰ Some reversible inhibitors bind so tightly to the enzyme that they essentially function as irreversible inhibitors.⁸⁶

Irreversible inhibitors have reactive groups that covalently, and thus permanently, bind the enzyme. They display time-dependent inhibition because the amount of active enzyme at a given concentration of irreversible inhibitor will be different depending on how long the inhibitor is preincubated with the enzyme. Because the enzyme will never be reactivated, inhibition lasts until new enzyme is synthesized.⁸⁶ One measure of inhibitor potency is the inhibitory concentration, K_i, the concentration of the inhibitor that produces 50% inhibition of the enzyme. The more potent the inhibitor, the lower the value.¹⁰⁴ Values below 1.0 μmol/L are regarded as potent.⁸² The azole antifungals are very potent, with K_i values of 0.02 μmol/L.¹⁰⁴

The impact of an inhibitor is also affected by the fraction of the substrate that is biotransformed by the inhibited, target enzyme. The inhibition of a CYP enzyme will have little impact if the enzyme only metabolizes a fraction of the affected xenobiotic.⁸² Conversely, xenobiotics that are primarily metabolized by a single CYP enzyme are more susceptible to interactions.⁸⁶ Simvastatin is biotransformed primarily by CYP3A4. The potent and specific CYP3A inhibitor itraconazole prevents its metabolism, resulting in an increased risk of rhabdomyolysis.¹

Specific CYP Enzymes

CYP1A1 and 1A2

Whereas 1A1 is located primarily in extrahepatic tissue, 1A2 is a hepatic enzyme and is involved in the metabolism of 10% to 15% of xenobiotics.^13,66 They both are very inducible by polycyclic aromatic hydrocarbons, including those in cigarette smoke and charred food. They bioactivate several procarcinogens, including benzo[a]pyrene.⁵⁷ Xenobiotics activated by the CYP1 enzyme family in the GI tract are linked to colon cancer.⁷⁸ A xenobiotic interaction through CYP1A2 involves the addition of ciprofloxacin, a moderate inhibitor of CYP1A2, to a patient using tizanidine for muscle spasms. Oral administration of tizanidine, an α₂ adrenergic agonist, yields low serum concentration because of extensive first-pass metabolism through CYP1A2. With concurrent ciprofloxacin dosing, serum tizanidine concentration increases significantly, and the interaction causes a clinically significant hypotension.³⁷

CYP2B6

This enzyme metabolizes approximately 7% of xenobiotics with many overlapping substrates with other CYP enzymes, especially CYP3A4.^43,125 CYP2B6 comprises between 4% and 14% of total hepatic CYP enzymes and is polymorphic with 38 allele variations.^49,125 Cyclophosphamide and ifosfamide are commonly used nitrogen mustard class of chemotherapeutics that require bioactivation. Metabolism through CYP2B6 yields the therapeutic phosphamide mustard metabolite, whereas metabolism through CYP3A4 inactivates the compound. Preferential metabolism through the induction of CYP2B6 increases therapeutic efficacy of cyclophosphamide and ifosfamide.⁴³

CYP2C9 and 2C19

The CYP2 enzyme family, with its 60 CYP2C9 alleles and 35 CYP2C19 alleles, is one of the most polymorphic of the CYP enzymes families (Table 11–1).^30,49,114 The CYP2C9 enzyme is the most abundant enzyme of the CYP2C enzyme subfamily, which, with CYP2C19, comprises approximately 10% to 20% of the CYP enzymes in the liver and is involved in 15% to 20% of all xenobiotic metabolism.^30,114 CYP2C9 biotransforms S-warfarin, the more active isomer of warfarin. Warfarin is one of the most commonly reported causes of adverse drug events. There is an association between slow metabolism and an increased risk of bleeding in patients taking warfarin.^16,97

CYP2D6

This enzyme is of historical significance because the exploration of differences in pharmacokinetics among individuals taking the antihypertensive debrisoquine led to the discovery of CYP2D6 and marked the beginning of pharmacogenetics.⁴⁸ Debrisoquine through its effects on blood pressure acts as a probe molecule for enzymatic variations of CYP2D6. In fact, CYP2D6 is highly polymorphic with more than 109 allelic variants.⁴⁹ Twenty-five percent of pharmaceuticals, including 50% of the commonly used antipsychotics and antidepressants, are substrates for CYP2D6.^42,48,78,83 Because CYP2D6 is the only drug-metabolizing CYP enzyme that is noninducible, the polymorphisms are the primary reason for the substantial interindividual variation in enzyme activity.⁵⁰ CYP2D6 enzyme is strongly inhibited by common antidepressants such as bupropion, fluoxetine, and paroxetine. Between inhibitors and poor metabolizers, opioid substrates such as codeine, tramadol, and hydrocodone that are bioactivated through CYP2D6 have less clinical efficacy.⁴² Ultrarapid metabolizers have higher incidences of postoperative and chemotherapy-induced vomiting when given ondansetron, a commonly used antiemetic metabolized by CYP2D6.⁹

CYP2E1

This enzyme comprises 7% of the total CYP enzyme content in the human liver.⁷⁹ It metabolizes small organic compounds, including ethanol, carbon tetrachloride, and halogenated anesthetics.¹⁰⁴ It also biotransforms low-molecular-weight xenobiotics, including benzene, acetone, and N-nitrosamines.¹⁰⁴ Some of its substrates are procarcinogens, which are bioactivated by CYP2E1.⁷⁸ The assessment for a relationship between CYP2E1 and cancer is intense because many of its substrates are environmental xenobiotics. The induction of CYP2E1 is associated with increased liver injury by reactive metabolites of carbon tetrachloride and vinyl chloride (Chap. 21).³⁶ During the metabolism of substrates that include carbon tetrachloride, ethanol, acetaminophen (APAP), aniline, and N-nitrosodimethylamine, CYP2E1 actively produces free radicals and other reactive metabolites associated with adduct formation and lipid peroxidation (Chaps. 9 and 33).¹⁷ CYP2E1 is inhibited by acute elevations of ethanol, an effect illustrated by the acute administration of ethanol inhibiting the metabolism of APAP. The chronic ingestion of ethanol hastens its own metabolism through CYP2E1 induction.³⁶

CYP3A4

CYP3A4 is the most abundant CYP in the human liver, comprising 40% to 55% of the mass of hepatic CYP enzymes.^13,30 The CYP3A4 enzyme is the most common one found in the intestinal mucosa and is responsible for much first-pass drug metabolism.¹³ It is involved in the biotransformation of 50% to 60% of all pharmaceuticals.^79,127 It has broad substrate specificity because it accommodates large lipophilic substrates and can adopt multiple conformations. It can even simultaneously fit 2 relatively large compounds (ketoconazole, erythromycin) in its active site.⁹⁶

Methadone, extensively metabolized by CYP3A4, is associated with many examples of adverse drug interactions.⁵⁴ Torsade de pointes due to the methadone dose–related prolongation of the QT_C interval is reported in patients who take methadone in addition to CYP3A4 inhibitors including ciprofloxacin,⁷⁵ itraconazole,⁸¹ and the antiretrovirals atazanavir and ritonavir.²⁹ Ketoconazole inhibits CYP3A4, causing a 15- to 72-fold increase in serum concentrations of terfenadine, which also causes torsade de pointes.⁷⁸ Bioflavonoids in grapefruit juice decrease metabolism of some substrates by 5- to 12-fold.^13,78 The CYP3A4 enzyme does not exhibit significant genetic polymorphism; however, there are large interindividual variations in enzyme concentrations that can affect metabolic rates.¹²⁷

Phase II Biotransformation Reactions

Phase II biotransformation reactions are synthetic, catalyzing the conjugation of products of phase I reactions or xenobiotics with endogenous molecules that are generally hydrophilic. Conjugation usually terminates the pharmacologic activity of xenobiotics and greatly increases their water solubility and excretability.^70,108,124 Conjugation occurs most commonly with glucuronic acid, sulfates, and glutathione. Less common phase II reactions include conjugation with amino acids such as glycine, glutamic acid, and taurine as well as acetylation and methylation.

Glucuronidation is the most common phase II synthesis reaction.⁷⁰ Glucuronyl-transferase has relatively low substrate affinity, but it has high capacity at higher substrate concentrations.¹⁰⁸ The glucuronic acid, donated by uridine diphosphate glucuronic acid, is conjugated with the nitrogen, sulfhydryl, hydroxyl, or carboxyl groups of substrates. Smaller conjugates usually undergo renal elimination, whereas larger ones undergo biliary elimination.⁷⁰ The process of glucuronidation is reversible via β-glucuronidases. Although the liver has an overall low concentration of β-glucuronidases, the gut flora have high β-glucuronidase activity. Cleavage of the glucuronide metabolite enhances the lipophilicity and thus reabsorption through intestinal walls. This process is termed enterohepatic circulation, which decreases elimination and prolong toxicity from xenobiotics such as carbamazepine and phenobarbital.^3,124 Multiple-dose activated charcoal provides therapeutic benefit to break enterohepatic circulation (Antidotes in Depth: A1).

Sulfation complements glucuronidation because it is a high-affinity but low-capacity reaction that occurs primarily in the cytosol. For example, the affinity of sulfate for phenol is very high (the K_m is low), so that when low doses of phenol are administered, the predominant excretion product is the sulfate ester. Because the capacity of this reaction is readily saturated, glucuronidation becomes the main method of detoxification when high doses of phenol are administered.^70,124 Sulfate conjugates are highly ionized and very water soluble.

Glutathione S-transferases are important because they catalyze the conjugation of the tripeptide glutathione (glycine-glutamate-cysteine, or GSH) with a diverse group of reactive, electrophilic metabolites of phase I CYP enzymes. The reactive compounds initiate an attack on the sulfur group of cysteine, resulting in conjugation with GSH that detoxifies the reactive metabolite. Of the 3 phase II reactions addressed, hepatic concentrations of glutathione by far account for the greatest amount of cofactors used. Although intracellular glutathione is difficult to deplete, when it does occur, severe hepatotoxicity often follows.¹²⁴ Some GSH conjugates are directly excreted. More commonly, the glycine and glutamate residues are cleaved and the remaining cysteine is acetylated to form a mercapturic acid conjugate that is readily excreted in the urine. A familiar example of this detoxification is the avid binding of N-acetyl-p-benzoquinoneimine (NAPQI), the toxic metabolite of APAP, by glutathione.^5,12

As with the CYP enzymes, many phase II enzymes are inducible. For example, UDP-glucuronosyltransferase, which performs glucuronidation, is inducible via the PXR, CAR, and AhR nuclear receptors after binding with rifampin, phenobarbital, and PAHs, respectively. Its activity varies 6- to 15-fold in liver microsomes.¹¹³ Similarly, UDP-glucuronosyltransferases are inhibited by xenobiotics such as amitriptyline, indinavir, phenylbutazone, and quinidine.^63,111 Inhibition of glucuronidation exacerbates hyperbilirubinemia in patients with underlying Gilbert syndrome (reduced UDP-glucuronosyltransferase activity on bilirubin).¹¹¹ Although inhibition of a high-capacity phase II metabolism step has theoretical implications and potential drug–drug interactions, the clinical implications are not well-studied.

Membrane Transporters

Although the focus on drug disposition has traditionally been on biotransformation, membrane transporter proteins greatly impact drug disposition.⁴⁵ Transporter proteins mediate the cellular uptake and efflux of endogenous compounds and xenobiotics.¹²⁸ Their baseline physiologic role is to transport sugars, lipids, amino acids, and hormones so as to regulate cellular solute and fluid balance. Biotransformation cannot occur unless xenobiotics are taken up into the cells via transport proteins. After xenobiotics have undergone phase I and II metabolism, the metabolites undergo transport protein mediated efflux from the cell, an action that has been called phase III metabolism.⁵²

Xenobiotic absorption, compartmentalization, and elimination are facilitated or prevented by the transport proteins.⁵⁸ In the apical (luminal) membrane of the intestinal enterocytes, P-glycoprotein, part of the ABC family of transmembrane proteins, is important because it can actively extrude xenobiotics back into the intestinal lumen and effectively decreases absorption.¹⁴ The degree of phenotypic expression of P-glycoprotein affects the bioavailability of many xenobiotics, including paclitaxel, digoxin, and protease inhibitors.⁴⁵ Transporter-mediated uptake of xenobiotics also occurs from the portal venous blood into the hepatocytes or from renal filtrate back into proximal tubular cells. α-Amanitin uses the sodium-dependent bile acid transporter, Na⁺-taurocholate cotransporting polypeptide, to enter hepatocytes, where it inhibits RNA-polymerase II and mediates cellular toxicity.⁴⁰ Alternatively, transport proteins decrease xenobiotic entry into specific cells or compartments. P-glycoprotein in endothelial cells of the blood–brain barrier prevent CNS entry of substrate xenobiotics. Finally, transport proteins mediate xenobiotic elimination via hepatocyte efflux transporters that move biotransformed xenobiotics into bile and via efflux from renal proximal tubular cells into urine.^14,45,128 Organs important for drug disposition have multiple transporters that have overlapping substrate capabilities, a redundancy that enhances protection.⁴⁵

As with biotransformation enzymes and nuclear receptors, membrane transporters are also inhibited or induced. Digoxin, a high-affinity substrate for P-glycoprotein, has increased bioavailability when administered with P-glycoprotein inhibitors such as clarithromycin or atorvastatin.⁴⁵ Loperamide is a substrate for P-glycoprotein that limits its intestinal absorption or CNS entry. Coadministration with quinidine, a P-glycoprotein inhibitor, or large dose ingestions (70–200 mg daily) that overwhelm the P-glycoprotein capacity result in increased opioid CNS effects of loperamide.^24,45 As with the biotransformation enzymes, polymorphisms exist for membrane transporters. However, the clinical significance of these is not clear.¹⁹

MECHANISMS OF CELLULAR INJURY

Ideally and commonly, potentially toxic metabolites produced by phase I reactions are detoxified during phase II reactions. However, detoxification does not always occur. This section reviews mechanisms of cellular injury related to xenobiotic biotransformation.

Synthesis of Toxins

Sometimes a xenobiotic is mistaken for an endogenous substrate by synthetic enzymes that biotransform it into an injurious compound. The incorporation of the rodenticide fluoroacetate into the citric acid cycle is an example of this mechanism of toxic injury (Fig. 11–3).⁹⁰ Another example is illustrated by analogs of purine or pyrimidine bases that are phosphorylated and inserted into growing DNA or RNA chains, resulting in mutations and disruption of cell division. This mechanism is used therapeutically with 5-fluorouracil (5-FU), an antitumor pyrimidine base analog. When phosphorylated to 5-fluoro-dUTP and incorporated into growing DNA chains, it causes structural instability of the cellular DNA and inhibits tumor growth.⁹⁴

Injury by Metabolites of Biotransformation

Many toxic products result from metabolic activation (Table 11–2).⁵³ The CYP enzymes most associated with bioactivation are 1A1, 1B1, 2A6, and 2E1, whereas 2C9 and 2D6 yield little toxic activation.³⁹

Highly reactive metabolites exert damage at the site where they are synthesized; reacting too quickly with local molecules to be transported elsewhere. This commonly occurs in the liver, the major site of biotransformation of xenobiotics (Chap. 21).^38,102 However, metabolism in the lungs, skin, kidneys, GI tract, and nasal mucosa also create toxic metabolites that cause local injury.^12,61 Overdoses of APAP lead to excessive hepatic CYP2E1 production of the highly reactive, semiquinoneimine, electrophile NAPQI, which initiates a damaging covalent bond with hepatocytes (Chap. 33).⁵ Acute renal tubular necrosis also occurs in patients with overdose of APAP, attributed to its biotransformation to NAPQI by prostaglandin H synthase within renal tubular cells.^28,118 Other toxic metabolites cause systemic toxicity. Dapsone (4,4’-diphenyldiaminosulfone) is a medication used in the treatment of Pneumocystis jiroveci pneumonia, malaria, and leprosy. Dapsone is metabolized primarily by CYP2C19 with contributions from CYP2B6, CYP2D6, and CYP3A4 to N-hydroxy metabolites such as dapsone hydroxylamine and monoacetyl hydroxylamine, which induce methemoglobinemia and cause hemolytic anemia.^6,33 These metabolites have a prolonged elimination half-life of approximately 30 hours and cause the well-described prolonged methemoglobinemia associated with dapsone.⁶

Monoamine oxidases (MAOs) are mitochondrial enzymes present in many tissues. They oxidize many amines, including dopamine, epinephrine, and serotonin, and xenobiotics such as primaquine and haloperidol. The metabolic activity of MAOs was responsible for the outbreak of parkinsonism associated with the use of methylphenyltetrahydropyridine (MPTP), an unintended by-product of attempts to synthesize a “designer” analog of meperidine, methylphenylpropionoxypiperidine (MPPP). After crossing the blood–brain barrier, MPTP is biotransformed by MAO in glial cells to methylphenyldihydropyridine (MPDP⁺), which is converted to the l-methyl-4-phenylpyridinium cation (MPP⁺). The MPP⁺ is subsequently taken up by specific dopamine transport systems into dopaminergic neurons in the substantia nigra, resulting in inhibition of oxidative phosphorylation and subsequent neuronal death.³⁵

Free Radical Formation

Within an atom, it is energetically favorable for electrons to exist in pairs or as a part of a chemical bond. An element or compound with an unpaired electron, called a radical or free radical, is highly reactive and short-lived. It rapidly seeks other species in order to obtain another electron. Radicals include the superoxide anion O2•-, which is produced by adding an electron to O₂, and the highly reactive hydroxyl radical HO•, which is produced by splitting the H₂O₂ molecule into 2 equal halves. The H₂O₂ molecule itself is reactive and is also associated with injury. The superoxide and hydroxyl radicals react with other molecules in order to stabilize themselves; however, by taking an electron in order to do so, they generate new free radical species, potentially initiating a chain reaction. Because the production of radicals occurs continually, the human body has developed effective defense mechanisms against these reactive molecules. However, some xenobiotics promote the formation of reactive oxidant species to the extent that antioxidant mechanisms are overwhelmed, a condition called oxidative stress. Oxidizing species are called such because, by reducing themselves by taking away electrons, they oxidize the species from which they took the electrons.⁸

Although oxidative stress results in oxidative damage to nucleic acids and proteins, other targets exist such as polyunsaturated fatty acids (PUFAs) in cellular membranes, resulting in lipid peroxidation (the oxidative destruction of lipids). This attack removes the particularly reactive hydrogen atom, with its lone electron, from a methylene carbon of a PUFA, leaving an unpaired electron and causing the formation of a lipid radical. This lipid radical attacks other PUFAs, initiating a chain reaction that destroys the cellular membrane. Membrane degradation products initiate inflammatory reactions in the cells, resulting in further damage.^8,102

Molecular oxygen (O₂) has a lone pair of electrons in its orbit. Because oxygen is a relatively weak univalent electron acceptor (and most organic molecules are weak univalent electron donors), oxygen cannot efficiently oxidize amino acids and nucleic acids. However, the unpaired electrons of O₂ readily interact with the unpaired electrons of transition metals and organic radicals. Metals frequently catalyze the creation of oxygen free radicals. Fig. 11–4 reflects the typical steps in the formation of a hydroxyl radical. The damaging effects of the free radicals are decreased by reaction with antioxidants such as ascorbate, tocopherols, and glutathione.⁷⁰ Deficiencies of antioxidants, especially glutathione, are associated with increased oxidative damage. Free radicals are also neutralized by several enzymes, including peroxidase, superoxide dismutase, and catalase.

The ethanol-inducible CYP2E1 enzyme produces significant amounts of superoxide and peroxide free radicals. In the presence of iron, these hydroxyl free radicals readily initiate lipid peroxidation. This was studied extensively in models of the metabolism of carbon tetrachloride, ethanol, and APAP.²³ The formation of free radicals is implicated in the pulmonary injury caused by paraquat, the myocardial injury caused by doxorubicin, and the hepatic injury caused by carbon tetrachloride.^73,91 Paraquat reacts with NADPH to form a pyridinyl free radical, which, in turn, reacts with oxygen to generate the superoxide anion radical. Doxorubicin is metabolized to a semiquinone free radical in the cardiac mitochondria, which, in the presence of oxygen, forms a superoxide anion radical that initiates myocardial lipid peroxidation.⁷³ Carbon tetrachloride (CCl₄) is metabolized to the trichloromethyl radical (•CCl₃) that binds covalently to cellular macromolecules. In the presence of oxygen, this is converted to the trichloromethylperoxyl radical (•CCl₃O₂) that can initiate lipid peroxidation (Fig. 11–5).⁹² (Chap. 105 offers a more extensive discussion.)

CRITICAL BIOCHEMICAL PATHWAYS AND XENOBIOTICS THAT AFFECT THEM

Energy metabolism is the foundation of cellular function. It provides high-energy fuel, predominantly in the form of ATP, for all energy-dependent cellular processes such as synthesis, active transport, and maintenance of electrolyte balance and membrane integrity. Numerous pathways interconnect glycogen, fat, and protein reserves in many tissues that store and retrieve ATP and glucose. The brain and red blood cells (RBCs) are entirely dependent on glucose for energy production, although other tissues can also use ketone bodies and fatty acids to synthesize ATP. Rapid cell death occurs if the production or use of ATP is inhibited; thus, the goal of many metabolic processes is the production and mobilization of cellular energy.

Catabolic pathways, those that break down molecules into smaller units enabling the production of cellular energy, include glycolysis, the citric acid cycle, and oxidative phosphorylation via the electron transport chain. Glycolysis produces small amounts of ATP through the anaerobic metabolism of glucose. Pyruvate, the end product of glycolysis, yields far more ATP when it is converted to acetyl-coenzyme A (acetyl-CoA) and “processed” in the citric acid cycle (Fig. 11–3). Fat and protein yield their energy through their conversion to acetyl-CoA and other intermediates of the citric acid cycle. The citric acid cycle and oxidative phosphorylation, via the electron transport chain, result in most ATP synthesis. Oxidative phosphorylation disposes of electrons or “reducing equivalents” and converts their energy to ATP. A lack of oxygen stops the electron transport chain and ATP production. Oxidative metabolism is highly energy efficient, producing 36 moles of ATP for each mole of glucose metabolized, compared to the 2 moles of ATP produced by anaerobic glycolysis. The following sections review the basics of cellular energy metabolism and several important xenobiotics that affect these critical metabolic functions (Table 11–3).^30,59

Glycolysis

Glycolysis is the first biochemical pathway in the metabolism of glucose. Other sugars enter this pathway after conversion to glycolytic intermediates (Fig. 11–6). The glycolytic process converts one molecule of glucose to 2 of pyruvate plus 2 molecules of ATP plus 2 molecules of NADH. Pyruvate may follow many paths. Under anaerobic conditions, the 2 pyruvate molecules produced from one glucose molecule are reduced by lactate dehydrogenase to 2 lactate molecules in an NADH-requiring step that regenerates NAD⁺. Thus, anaerobic glycolysis yields 2 molecules of lactate plus 2 molecules of ATP. When NAD⁺ and oxygen are available, pyruvate is transported from the cytosol into the mitochondria where it enters the citric acid cycle by being converted by pyruvate decarboxylase to acetyl-CoA (Fig. 11–3).^30,59 In energy-rich conditions, pyruvate is used for fatty acid synthesis, whereas in energy-poor situations, it is used for gluconeogenesis.

Arsenate (As⁵⁺) affects the glycolytic step where 3-phosphoglyceraldehydedehydrogenase (3-PGA) catalyzes the oxidation of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate, a reaction that preserves a high-energy phosphate bond used to synthesize ATP in the next step of glycolysis (Fig. 11–6).⁴⁶ Arsenate acts as an analog of phosphate at this step, resulting in an unstable arsenate intermediate that is rapidly hydrolyzed to 3-phosphoglycerate. While glycolysis continues, there is the loss of ATP synthesis because 1,3 biphosphoglycerate is not made.⁴⁶

Citric Acid Cycle

The citric acid cycle uses acetyl-CoA derived from glycolysis, fat, or protein to regenerate NADH from NAD⁺. The cycle is a major source of electrons (in the form of NADH) and is critical to the aerobic production of ATP (Fig. 11–3). Each acetyl-CoA molecule that is oxidized within the citric acid cycle ultimately forms one molecule each of CO₂ and guanosine triphosphate (GTP), and more importantly, 3 molecules of NADH and one molecule of reduced flavin adenine dinucleotide (FADH₂). Both NADH and FADH₂ enter the electron transport chain to generate ATP via oxidative phosphorylation (details below). In addition, the citric acid cycle provides important intermediates for amino acid synthesis and for gluconeogenesis.⁵⁹

Various xenobiotics inhibit the citric acid cycle. The rodenticides, sodium fluoroacetate and fluoroacetamide, are combined with coenzyme A, CoASH, to create fluoroacetyl CoA (FAcCoA). The FAcCoA substitutes for acetyl CoA, entering the citric acid cycle by condensation with oxaloacetate to form fluorocitrate. Fluorocitrate metabolism results in a metabolite that blocks aconitase within the cycle, resulting in citrate accumulation and the termination of oxidative metabolism (Fig. 11–3) (Chap. 113).

Thiamine is an important cofactor for 2 citric acid cycle enzymes: the conversion of pyruvate to acetyl-CoA by pyruvate decarboxylase and for the conversion of α-ketoglutarate to succinyl-CoA by α-ketoglutarate dehydrogenase (Fig. 11–3).³⁰ The life-threatening effects of thiamine deficiency are likely related to impairment of these enzyme functions (Antidotes in Depth: A27). Chronic alcoholics have poor nutrition intake with impaired thiamine absorption. Thiamine deficiency damages cholinergic neurons in the basal forebrain leading to a syndrome of diencephalic amnesia and contributes to the pathophysiology of Wernicke encephalopathy and the Korsakoff psychosis. These cholinergic neurons have high energy demands and are more sensitive to inhibitions of glucose metabolism through thiamine-dependent enzymes.⁷⁶

The Electron Transport Chain

The electron transport chain is the location where the “phosphorylation” of oxidative phosphorylation occurs. Oxidative phosphorylation is the creation of high-energy bonds by phosphorylation of adenosine diphosphate (ADP) to ATP, “coupled” to the transfer of electrons from reduced coenzymes to molecular oxygen via the electron transport chain. The success of aerobic metabolism requires the disposal of electrons within NADH and FADH₂, generated by oxidative metabolism within glycolysis and the citric acid cycle. The electron transport chain consists of a series of cytochrome–enzyme complexes within the inner mitochondrial membrane (Fig. 11–3). Within these complexes, NADH is split into NAD⁺ + H⁺ plus 2 electrons at complex I at the beginning of the chain whereas FADH₂ is split into FAD + 2H⁺ plus 2 electrons at complex II. These reactions have 2 results. First, the regenerated NAD⁺ and FAD are recycled back to the citric acid cycle, enabling oxidative metabolism to continue. Second, these actions provide the energy required to pump protons (H⁺) from the mitochondrial matrix into the intermembrane space. This action causes the matrix to become relatively alkaline compared to the now acidified intermembrane space and the creation of a proton gradient across the inner mitochondrial membrane (mitochondrial membrane potential, Δψ_m ~ 150–180 mV).⁸⁷ The final step in the electron flow of oxidative phosphorylation is the reduction of molecular oxygen to water by complex IV, cytochrome a-a₃ (Fig. 11–3).^30,59 This H⁺ gradient provides the energy needed to create the high-energy bonds of ATP at complex V (ATP synthase).

In total, aerobic glucose metabolism generates 36 moles of ATP. Glycolysis contributes 2 moles of ATP, 2 moles of NADH, and 2 moles of pyruvate. Pyruvate dehydrogenase converts pyruvate to acetyl-CoA while generating another 2 moles of NADH per mole of glucose. The citric acid cycle uses acetyl-CoA to generate 6 moles of NADH, 2 moles of FADH₂, and 2 mol of GTP per mole of glucose. Each mole of NADH via the electron transport chain produces 3 moles of ATP. Flavin adenine dinucleotide donates electrons later in the chain, so it only produces 2 moles of ATP. The entire process generates 2 moles of ATP, 10 moles of NADH (30 moles of ATP), and 2 moles of FADH₂ (4 moles of ATP) for a total of 36 moles of ATP and 2 moles of GTP per mole of glucose.

Mitochondria oxidize substrates, consume oxygen, produce CO₂, and make ATP. Xenobiotics that interrupt oxidative phosphorylation impair ATP production either by inhibiting specific electron chain complexes or by acting as “uncouplers.” Both of these mechanisms result in rapid depletion of cellular energy stores, followed by failure of ATP-dependent active transport pumps, loss of essential electrolyte gradients, and increases in cell volume.⁵⁹

Inhibitors of specific cytochromes block electron transport and cause an accumulation of reduced intermediates proximal to the site of inhibition. This stops the regeneration of oxidized substrates for the citric acid cycle, particularly NAD⁺ and FAD, further impairing oxidative metabolism. Cyanide, carbon monoxide, and hydrogen sulfide block the cytochrome a-a₃–mediated reduction of O₂ to H₂O. The very dramatic clinical effects of these exposures illustrate the importance of aerobic metabolism (Chap. 123). Other xenobiotics are less commonly associated with inhibition of the electron transport chain (Table 11–3).^110,116

Severe metabolic acidosis is a clinical manifestation of xenobiotics that inhibit aerobic respiration. Lactate (the base form of lactic acid) accumulates during anaerobic respiration but is only a surrogate marker for metabolic acidosis associated with the impairment of oxidative metabolism.⁶⁰ The process of anaerobic glycolysis from glucose to pyruvate and subsequently to lactate is H⁺ neutral (Fig. 11–7, Equation 1). The H⁺ excess results from ATP hydrolysis by energy-requiring cellular processes. The hydrolysis of a high-energy bond in ATP yields ADP, phosphate, H⁺, and energy (Fig. 11–7, Equation 2). In total, 1 mole of glucose via anaerobic glycolysis yields 2 moles of lactate and 2 moles of H⁺ (Fig. 11–7, Equation 3). Therefore, the hydrolysis of ATP under anaerobic conditions contributes to metabolic acidosis rather than the dissociation of lactic acid to lactate.⁶⁸ Conversely during aerobic respiration and oxidative phosphorylation, the process of ATP generation via ATP synthase consumes the H⁺ gradient created by the electron transport chain and largely balances H⁺ production during ATP hydrolysis.

Xenobiotics that uncouple oxidative phosphorylation, like inhibitors of the electron transport chain, reduce ATP synthesis. However, with uncouplers, protons continue to be pumped into the intermembrane space, electrons continue to flow down the chain to reduce oxygen, and substrate consumption continues. Uncouplers dissipate the proton gradient across the mitochondrial inner membrane by allowing the protons to cross back into the mitochondrial matrix. Because it is the proton gradient that drives the production of ATP at complex V, ATP production is reduced. Thus, oxygen consumption is “uncoupled” from ATP production. The redox energy created by electron transport that cannot be coupled to ATP synthesis is released as heat. Various xenobiotics uncouple ATP synthesis (Table 11–3). A classic one is dinitrophenol, used as an herbicide and as a weight-loss product (Chap. 40). Xenobiotics that are capable of carrying hydrogen ions across membranes are generally lipophilic weak acids. These xenobiotics must have an acid-dissociable group to carry the proton and a bulky lipophilic group to cross a membrane.⁶² Dinitrophenol is able to carry its proton from the cytosol into the more alkaline mitochondrial matrix where it dissociates, acidifying the matrix and destroying the proton gradient across the inner mitochondrial membrane. Interestingly, the phenolate anion of dinitrophenol is relatively lipophilic and can cross back out to the cytosol where it gains a new proton and starts the process over again. Long-chain fatty acids uncouple oxidative phosphorylation by a similar mechanism.^62,116 Fatal exposures to dinitrophenol and to pentachlorophenol, a wood preservative, are associated with severe hyperthermia attributed to heat generation by uncoupled oxidative phosphorylation.⁸⁹ The hyperthermia and acidosis associated with severe salicylate poisoning are attributed to its uncoupling of oxidative phosphorylation.¹⁰⁷

Hexose Monophosphate Shunt

The hexose monophosphate shunt provides the only source of cellular NADPH. Nicotinamide adenine dinucleotide phosphate is used in biosynthetic reactions, particularly fatty acid synthesis, and is an important source of reducing power for the maintenance of sulfhydryl groups that protect the cell from free radical injury.^10,47 As noted earlier, G6PD is a key enzyme in the pathway (Fig. 11–8). Reduced glutathione, which is quantitatively the most important antioxidant in cells, depends on the availability of NADPH. Red blood cells are especially vulnerable to deficiency of NADPH, which results in hemolysis during oxidative stress.

Another manifestation of oxidative stress in RBCs is the oxidation of the iron in hemoglobin from Fe²⁺ to Fe³⁺, producing methemoglobin that occurs both spontaneously and as a response to xenobiotics such as nitrites and aminophenols. Because most reduction of methemoglobin is done by NADH-dependent methemoglobin reductase, which is not deficient in persons who lack G6PD, such persons do not develop methemoglobinemia under normal circumstances. However, when oxidative stress is severe and methemoglobinemia develops, people who have G6PD deficiency have limited ability to use the alternative NADPH-dependent methemoglobin reductase (Chap. 124).¹¹²

Gluconeogenesis

Gluconeogenesis facilitates the conversion of amino acids and intermediates of the citric acid cycle to glucose. It is an important source of glucose during fasting and enables maintenance of glycogen stores. The brain is a major consumer of glucose from both dietary intake and from gluconeogenesis. Other tissues such as the testes, erythrocytes, and kidney medulla rely exclusively on glucose for energy production.⁵⁹ Gluconeogenesis occurs primarily in the liver but also in the kidney. Most of the steps in the synthesis of glucose from pyruvate are simply the reverse of glycolysis, with 3 irreversible exceptions: (1) the conversion of glucose-6-phosphate to glucose; (2) the conversion of fructose-1,6-diphosphate to fructose-6-phosphate; and (3) the synthesis of phosphoenolpyruvate from pyruvate. The synthesis of phosphoenolpyruvate from pyruvate is especially complex. Pyruvate is first converted to oxaloacetate within the mitochondria; then to malate, which is transported out of the mitochondria and converted in the cytosol back to oxaloacetate; and then to phosphoenolpyruvate (Fig. 11–9). Certain amino acids—notably alanine, glutamate, and aspartate—are readily converted to citric acid cycle intermediates and can be used in the synthesis of glucose through this cycle.⁵⁹ Glycerol, produced by the breakdown of triglycerides in adipose tissue, is another substrate for gluconeogenesis.

The regulation of gluconeogenesis is opposite to that of glycolysis, stimulated by glucagon and catecholamines but inhibited by insulin. Gluconeogenesis requires the cytosolic NAD⁺ and mitochondrial NADH. It is impaired by processes that increase the cytosol-reducing potential as measured by the cytosol NADH/NAD⁺ ratio (see discussion below).

A number of xenobiotics impair gluconeogenesis, resulting in hypoglycemia when glycogen stores are depleted (Table 11–3). Hypoglycin A, an unusual amino acid found in unripe ackee fruit that is the cause of Jamaican vomiting sickness, and its metabolite, methylenecyclopropylacetic acid (MCPA), found in lychee fruit, produce profound hypoglycemia.^98,105 Methylenecyclopropylacetic acid indirectly inhibits gluconeogenesis by blocking the oxidation of long-chain fatty acids, an important source of NADH in mitochondria. It also inhibits the metabolism of several glycogenic amino acids, including leucine, isoleucine, and tryptophan, and blocks their entrance into the citric acid cycle. Methylenecyclopropylacetic acid also prevents the transport of malate out of the mitochondria.^93,105,106 Hypoglycemia often occurs in fasting patients with elevated ethanol concentrations.^4,32,61 This is likely a result of the impairment of gluconeogenesis by the increased cytosolic NADH:NAD⁺ ratio associated with the metabolism of ethanol. This inhibits the 2 cytosolic steps that require NAD⁺—the conversions of lactate to pyruvate and of malate to oxaloacetate.^2,61

Fatty Acid Metabolism

Fatty acid metabolism occurs primarily in hepatocytes. Fatty acids mobilized in adipose tissue enter hepatocytes by passive diffusion. Fatty acid synthesis is stimulated by insulin and inhibited by glucagon and epinephrine. Acetyl-CoA is the primary building block of free fatty acids (FFAs). In energy-depleted cells, fatty acids are combined with glycerol phosphate to form triacylglycerol (triglycerides), the first step in the synthesis of fat for storage. Hepatic triglycerides are bound to lipoprotein to form very-low-density lipoprotein and then transported and stored in adipocytes. When hepatocytes are energy depleted, triglycerides are broken down to FFAs and glycerol. This process is suppressed by insulin but supported by glucagon or epinephrine. Free fatty acids undergo β-oxidation in the mitochondria, a process that breaks the FFA into acetyl-CoA molecules that can then enter the citric acid cycle. Free fatty acids require activation before transport into the mitochondria. This is accomplished by acyl-coenzyme A (acyl-CoA) synthetase, which adds a CoA group to the FFA in an energy-dependent synthetic reaction. These are transported into the mitochondria by a process that utilizes cyclical binding to carnitine, a “carnitine shuttle” (Fig. 11–3). Once inside the mitochondria, FFAs are converted to acetyl-CoA by β-oxidation, involving the sequential removal of 2-carbon fragments, each time acting at the second carbon (the β carbon) position of the fatty acid. Each 2-carbon mole removed from the FFA produces one NADH and one FADH₂, which are oxidized in the electron transport chain, and 1 mole of acetyl-CoA, which enters the citric acid cycle. This process produces 1.3 times more ATP per mole of carbon metabolized than does the oxidative metabolism of glucose or other carbohydrates.⁵⁹

Many xenobiotics interrupt fatty acid metabolism at various steps, resulting in accumulation of triglycerides in the liver (Table 11–3; Fig. 11–10). The mechanisms of disruption of fatty acid metabolism are poorly defined.¹⁸ Some xenobiotics, including ethanol, hypoglycin A, MCPA, and nucleoside analogs, inhibit β-oxidation, at least indirectly, through effects on NADH concentrations. Protease inhibitors are associated with a syndrome of peripheral fat wasting, central adiposity, hyperlipidemia, and insulin resistance.

The condition of alcoholic ketoacidosis is related in part to inhibition of gluconeogenesis in the alcoholic patient and in part to an exuberant response to nutritional needs by the fatty acid machinery. In a chronic alcohol-users, decreased intake of carbohydrates and impaired thiamine absorption stimulates a starvation response with increases in serum glucagon, cortisol, growth hormone, and epinephrine concentrations, and decreases in serum insulin. With continued ethanol intake, the metabolism of ethanol via oxidation increases the NADH:NAD⁺ ratio, which inhibits hepatic gluconeogenesis. When the need for carbohydrate is not met by gluconeogenesis, lipolysis, which is normally inhibited by insulin, is intensified and fatty acid mobilization progresses. The increased mitochondrial NADH:NAD⁺ ratio favors the production of β-hydroxybutyrate over acetoacetate, its oxidized form. The administration of fluids, dextrose, and thiamine to the alcoholic patient leads to correction of this process (Chap. 76).⁷¹

Protein and Nitrogen Metabolism

Human nitrogen consumption occurs through dietary intake of free amino acids, polypeptides, and ammonia (via intestinal splitting of urea and amino acids). Amino acids are the basic building blocks of proteins and are built on a backbone of an amino group, a carboxylic acid group, and a middle α carbon attached to a variable group. The simplest amino acid is glycine, in which the variable group is a hydrogen. Of the 20 major amino acids involved in protein synthesis, 10 are considered essential and only available through dietary intake. The other amino acids can be synthesized by the body via other carbon- and nitrogen-containing molecules. As mentioned above, amino acids such as alanine, glutamate, and aspartate are available for use as citric acid cycle intermediates. Alanine is an important molecule for skeletal muscles for the elimination of nitrogen waste and for energy generation via the glucose-alanine cycle. Myocytes release alanine to the systemic circulation through which it goes to the liver. In the hepatocytes, alanine converts to pyruvate via alanine transaminase (ALT), releasing its nitrogen group to glutamate. Pyruvate then undergoes gluconeogenesis, and glucose is released back to circulation for myocytes and other cells to use.⁵⁹

Glutamine is the main storage molecule for total body nitrogen/ammonia. A single glutamine molecule contains 2 amino groups. The uptake of ammonia (NH₄⁺) into glutamine protects the body from the neurotoxic and alkalemic effects of free ammonia. The removal of an ammonia yields glutamate (a neurotransmitter), and a removal of a second ammonia yields α-ketoglutarate (a citric acid cycle substrate). As free ammonia is released from glutamine, hepatocytes facilitate the removal of free ammonia from the body via generation of urea by the urea cycle (Fig. 11–11). The activation step of the urea cycle combines NH₄⁺, bicarbonate, and 2 ATP molecules to create carbamoyl phosphate by carbamoyl phosphate synthetase-1 (CPS-1). This process requires an obligate cofactor of N-acetylglutamate, a product of glutamate and acetyl-CoA via N-acetylglutamate synthetase. Carbamoyl phosphate enters into the urea cycle through ornithine transcarbamylase (OTC), which catalyzes the addition of carbamoyl phosphate to ornithine to make citrulline. Citrulline is subsequently converted to arginosuccinate, arginine, and then back to ornithine. The final step of arginine to ornithine by arginase releases urea. Urea is highly water soluble, and it is renally eliminated from the body.⁵⁹

Inborn errors of metabolism involving the deficiencies of urea cycle enzymes lead to severe hyperammonemia and death if undiagnosed and/or untreated. X-linked OTC deficiency is the most common urea cycle disorder. Several xenobiotics can also induce secondary hyperammonemia by the inhibition of enzymes in the urea cycle (Table 11–4).²⁰ For example, valproic acid undergoes ω-oxidation to yield 2-propyl-4-pentenoate (4-en-VPA) and propionate in an overdose state or when secondary carnitine deficiencies prohibit adequate β-oxidation. Both 4-en-VPA and propionate decrease the generation of N-acetylglutamate, which effectively inhibits CPS-1 and subsequently the entire urea cycle. Hyperammonemia is a common clinical feature of valproic acid in overdose and occasionally in therapeutic use (Chap. 48).¹²²

SUMMARY

• Biotransformation is a complex process, involving numerous enzyme systems, that usually detoxifies xenobiotics; however, “metabolic activation” occurs.

• Phase I reactions add functional groups to lipophilic xenobiotics to enhance their reactivity, preparing them for further detoxification by synthetic phase II reactions that enhance hydrophilicity and renal elimination.

• The phase I cytochrome P450 (CYP) enzymes biotransform most xenobiotics; CYP3A4 is the most abundant one, metabolizing 50% to 60% of all pharmaceuticals.

• Cytochrome P450 enzyme activity is quite variable because of genetic polymorphisms—genetic changes that exist in at least 1% of the human population. Enzyme activity also varies because of induction and inhibition.

• The most common method of induction is a nuclear receptor–mediated increase in gene transcription. Inhibition of CYP enzymes is the most common cause of harmful drug–drug interactions, the most common type being competitive, where the substrate and inhibitor both bind the active site of the enzyme.

• Xenobiotic disposition is also effected by membrane transporter (MT) proteins that mediate the cellular uptake and efflux of xenobiotics.

• P-glycoprotein is an MT protein that mediates the efflux of xenobiotics from the apical (luminal) membrane of intestinal enterocytes.

Acknowledgment

Kathleen A. Delaney, MD, contributed to this chapter in previous editions.

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APPENDIX

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